Devices and methods for sample partitioning and analysis

ABSTRACT

The present disclosure provides a device that includes a base comprising a substrate having a first major surface, a pressure sensitive adhesive adhered to at least a portion of the first major surface, a polymeric cover film coupled to the substrate via the adhesive, a plurality of isolated closed compartments disposed between the substrate and the cover film, and an aqueous liquid disposed in two or more of the closed compartments. The cover film is a composite film comprising ethylene vinyl acetate copolymer, a linear copolymer of ethylene and a higher alkene, and a tackifier. Each compartment of the plurality is defined by a seal that prevents liquid communication with at least one other compartment of the plurality. Methods of using the device for partitioning a sample, for analyzing a sample, and for culturing a microorganism are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/039,638, filed Aug. 20, 2014, the disclosure of which isincorporated by reference in its entirety herein.

TECHNICAL FIELD

The invention relates to a method of analyzing, for instance,enumerating and/or detection of microorganisms or other biologicalmaterial in a liquid sample and a device adapted for use in such amethod.

BACKGROUND

Many industries need to detect and quantify biological material in asample, for instance, the determination of microbial concentration infood and water is an essential part of food and water quality testing.Similar demands arise from a multitude of industries including food,biotechnological, pharmaceutical, water treating industry, and also inmedical microbiological diagnostics, environmental and scientificresearch. Samples are commonly scrutinized to, for instance, monitormicrobial population in a production environment, in-process controls,post storage and also final product testing.

Classical methods for the examination of samples particularly liquidsamples typically demands incubation time or reaction time for analysis.Analysis may involve several different kinds of chemical, biochemical,physical or optical techniques and require many hours or even days forincubation and subsequent analysis. Reducing the time for quantitativeand qualitative analysis of samples is very essential for making rapiddecisions in quality and process control operations.

With regard to testing of aqueous biological samples, it is advantageousto partition the sample into aliquots so that the desired reaction orgrowth occurs and can be detected much more rapidly than the samereaction or growth in the original larger volume. Biological samplessuch as microbiological samples and molecular biology samples wouldoften require such partitioning, in order to be analyzed preciselyqualitatively and/or quantitatively.

Such methods and device envisage a series of tiny compartments which arefilled en masse with an aqueous sample. In each case, once filled andsealed, the desired reaction or growth occurs and can be detected muchmore rapidly than the same reaction or growth in the original largervolume.

SUMMARY

In general, the present disclosure relates to devices and method forpartitioning a liquid sample (e.g., an aqueous liquid sample). Inaddition, the present disclosure relates to a method of using thedevices to detect a microorganism and/or biomolecule in the liquidsample.

In one aspect, the present disclosure provides a quick, simple andconvenient device for partitioning of liquid sample into a large numberof smaller discrete volumes in an efficient, robust and economicalmanner.

The present disclosure further provides a pouch that can be used forpartitioning of liquid sample into a large number of smaller discretevolumes in an efficient, robust and economical manner. Advantageously,the pouch may be provided with nutrients and/or indicators disposedtherein for detecting microorganisms. The nutrients and/or indicatorsmay be provided as a dry powder coating, which is dissolved when aliquid (e.g., an aqueous liquid) is dispensed into the pouch. The pouchcan be used in a method to create a device in which the liquid ispartitioned into a plurality of isolated, closed compartments.

Advantageously, the present disclosure provides a device for easypartitioning of liquid wherein the device comprises two films, onecoated with a pressure sensitive adhesive and the other being deformableso as to conform to the shape of the desired partitions such that thesample is partitioned into small discrete units without permitting themixing of sample in the adjacent compartments.

In one aspect, the present disclosure provides a device that cancomprise a base comprising a substrate having a first major surface, apressure sensitive adhesive adhered to at least a portion of the firstmajor surface, a polymeric cover film coupled to the substrate via theadhesive, a plurality of closed compartments disposed between thesubstrate and the cover film, and an aqueous liquid disposed in two ormore of the closed compartments. The cover film can be a composite filmcomprising a polymer and a tackifier. The composite film can compriseethylene vinyl acetate copolymer, a linear copolymer of ethylene and ahigher alkene, and the tackifier. Each compartment of the plurality canbe defined by a seal that prevents liquid communication with anothercompartment of the plurality. The seal is formed by contact between thecover film and the pressure sensitive adhesive.

In another aspect, the present disclosure provides a device that cancomprise a base comprising a substrate having a first major surface, apressure sensitive adhesive adhered to at least a portion of the firstmajor surface, a cover film coupled to the substrate via the adhesive, aplurality of closed compartments disposed between the substrate and thecover film, and an aqueous liquid disposed in two or more of the closedcompartments. The cover film can have an elastic recovery of less thanor equal to 20%. Each compartment of the plurality can be defined by aseal that prevents liquid communication with another compartment of theplurality. The seal is formed by contact between the cover film and thepressure sensitive adhesive.

The device as disclosed in the present invention finds a variety ofapplications in molecular biology, biochemistry, biotechnology andmicrobiology applications.

Advantageously, the device of the present invention facilitates improvedtime-to-result and easily expanded testing capabilities including rapiddetection of microorganisms, molecular characterization, indicatororganism testing, single organism pathogen enrichment, most probablenumber (MPN) style testing formats, and high-throughput post-enrichmentbiochemical characterization coupled with a easy-to-use and time savingbenefit.

In yet another aspect, the present disclosure provides a method forpartitioning liquid. The method can comprise depositing a predefinedvolume of liquid between a substrate and a polymeric cover film whereinsaid substrate is coated with water-insoluble pressure sensitiveadhesive, and urging an external means against the cover film to bringdiscrete regions of the cover film in contact with the pressuresensitive adhesive of the substrate resulting in the partitioning of theliquid into a plurality of closed compartments disposed between thesubstrate and the cover film. The cover film can be a composite filmcomprising a polymer and a tackifier. The composite film can compriseethylene vinyl acetate copolymer, a linear copolymer of ethylene and ahigher alkene, and the tackifier.

In yet another aspect, the present disclosure provides a method forpartitioning liquid. The method can comprise depositing a predefinedvolume of liquid onto a first surface of a substrate such that theliquid sample is disposed between the first surface and a polymericcover film wherein said first surface is coated with water-insolublepressure sensitive adhesive, and urging an external means against thecover film to bring discrete regions of the cover film in contact withthe pressure sensitive adhesive of the substrate resulting in thepartitioning of the liquid into a plurality of closed compartmentsdisposed between the substrate and the cover film. The cover film canhave an elastic recovery of less than or equal to 20%.

According to another aspect, the present disclosure provides a methodfor analyzing a liquid sample for its quantitative and qualitativeaspects. The method can comprise depositing a liquid sample onto a firstsurface of a substrate such that the liquid sample is disposed betweenthe first surface and a polymeric cover film wherein said first surfaceis coated with water-insoluble pressure sensitive adhesive, urging anexternal means against the cover film to bring discrete regions of thecover film in contact with the pressure sensitive adhesive of thesubstrate resulting in the partitioning of the liquid into a pluralityof closed compartments disposed between the substrate and the coverfilm, and conducting a quantitative analysis or a qualitative analysisof at least one closed compartment of the plurality. The cover film canbe a composite film comprising a polymer and a tackifier. The compositefilm can comprise ethylene vinyl acetate copolymer, a linear copolymerof ethylene and a higher alkene, and the tackifier.

According to another aspect, the present disclosure provides a methodfor analyzing a liquid sample for its quantitative and qualitativeaspects. The method can comprise depositing a liquid sample onto a firstsurface of a substrate such that the liquid sample is disposed betweenthe first surface and a polymeric cover film wherein said first surfaceis coated with water-insoluble pressure sensitive adhesive, urging anexternal means against the cover film to bring discrete regions of thecover film in contact with the pressure sensitive adhesive of thesubstrate resulting in the partitioning of the liquid into a pluralityof closed compartments disposed between the substrate and the coverfilm, and conducting a quantitative analysis or a qualitative analysisof at least one closed compartment of the plurality. The cover film canhave an elastic recovery of less than or equal to 20%.

In another embodiment, the present disclosure provides a method for theisolation, detection, culturing and enrichment of microorganisms,including anaerobic and aerobic forms.

In any embodiment, the analysis for the detection and enumeration ofmicroorganisms is carried out by the approaches described herein whichallow for the use of water-soluble indicator species, and reduces oreliminate the need for the several dilutions typically required incurrent microbiological analyses.

In yet another aspect, the present disclosure provides a method forculturing an aerobic or an anaerobic microorganism. The method cancomprise mixing a sample with a liquid nutrient medium to render itliquefied, depositing the liquefied sample onto a first surface of asubstrate such that the liquid sample is disposed between the firstsurface and a polymeric cover film wherein said substrate is coated withwater-insoluble pressure sensitive adhesive, urging an external meansagainst the cover film to bring discrete regions of the cover film incontact with the pressure sensitive adhesive of the substrate resultingin the partitioning of the liquid into a plurality of closedcompartments disposed between the substrate and the cover film, andincubating the liquefied and partitioned sample, under conditions tofacilitate at least one cell division of said microorganism. The coverfilm can be a composite film comprising a polymer and a tackifier. Thecomposite film can comprise ethylene vinyl acetate copolymer, a linearcopolymer of ethylene and a higher alkene, and the tackifier.

In yet another aspect, the present disclosure provides a method forculturing an aerobic or an anaerobic microorganism. The method cancomprise mixing a sample with a liquid nutrient medium to render itliquefied, depositing the liquefied sample onto a first surface of asubstrate such that the liquid sample is disposed between the firstsurface and a polymeric cover film wherein said first surface is coatedwith water-insoluble pressure sensitive adhesive, urging an externalmeans against the cover film to bring discrete regions of the cover filmin contact with the pressure sensitive adhesive of the substrateresulting in the partitioning of the liquid into a plurality of closedcompartments disposed between the substrate and the cover film, andincubating the liquefied and partitioned sample, under conditions tofacilitate at least one cell division of said microorganism. The coverfilm can have an elastic recovery of less than or equal to 20%.

Another aspect of the present invention is to provide a kit forpartitioning a liquid sample into a plurality of discrete compartments.The kit can comprises a substrate having a first major surface with alayer of a water-insoluble pressure sensitive adhesive adhered thereto;and a composite cover film. The composite film can comprise ethylenevinyl acetate copolymer, a linear copolymer of ethylene and a higheralkene, and a tackifier.

Another aspect of the present invention is to provide a kit forpartitioning a liquid sample into a plurality of discrete compartments.The kit can comprises a substrate having a first major surface with alayer of a water-insoluble pressure sensitive adhesive adhered thereto;and a polymeric cover film having an elastic recovery of less than orequal to 20%.

In any of the above embodiments of the kit, the substrate and/or thecover film can be substantially planar. In any of the above embodimentsof the kit, the pressure sensitive adhesive can comprise siliconepolyurea. In any of the above embodiments of the kit, the substratefurther can comprise a secondary coating disposed on at least a portionof the adhesive. In any of the above embodiments of the kit, wherein aspacer element is coupled to the first major surface of the substrate.In any of the above embodiments of the kit, the cover film is attachedto the substrate; wherein the spacer element, if present, is disposedbetween the substrate and the cover film.

In yet another aspect, the present disclosure provides a kit. The kitcan comprise a substrate having a first major surface with a layer of awater-insoluble pressure sensitive adhesive adhered thereto, and acomposite polymeric film. The polymeric film can comprise ethylene vinylacetate copolymer, a linear copolymer of ethylene and a higher alkene,and a tackifier. The tackifier can be selected from a group consistingof a low molecular weight polyisobutene, polyterpenes, amorphouspolypropylene, and microcrystalline wax. In any embodiment of the kit,the composite polymeric film has an elastic recovery of less than orequal to 20%.

In yet another aspect, the present disclosure provides a kit. The kitcan comprise a substrate having a first major surface with a layer ofwater-insoluble pressure sensitive adhesive adhered thereto, and apolymeric film having an elastic recovery of less than or equal to 20%.

In any of the above embodiments of the kit, the substrate and/or thepolymeric film is substantially planar. In any of the above embodimentsof the kit, the substrate and/or the polymeric film is substantiallyflat. In any of the above embodiments of the kit, the pressure sensitiveadhesive comprises silicone polyurea. In any of the above embodiments ofthe kit, the substrate further comprises a secondary coating disposed onat least a portion of the adhesive. In any of the above embodiments ofthe kit, the secondary coating comprises a powdered nutrient and/or aplurality of glass bubbles. In any of the above embodiments of the kit,the secondary coating is substantially water-free. In any of the aboveembodiments of the kit, a spacer element is coupled to the first majorsurface of the substrate, as described hereinabove. In any of the aboveembodiments of the kit, the composite polymeric film is attached to thesubstrate, wherein the spacer element, if present, is disposed betweenthe substrate and the cover film.

As described herein, the present invention has several advantages.First, the articles and methods eliminate the use of device havingpreformed compartments, thereby permitting the operator to choose from avariety of potential partition configurations (e.g., the number ofcompartments, the volume of each compartment, the total volume ofsample) at the point of use. Second, use of microvolumes inmicrocompartments allows for a surprisingly rapid detection andenumeration of microorganisms in a liquid test sample. The invention isparticularly useful in MPN analysis of a liquid test sample for aparticular microorganism, such as E. coli or S. aureus. The inventionallows MPN analysis to be conducted conveniently in a single device, asopposed to separate tubes, and advantageously requires a substantivelyshorter incubation time to reach detectable microorganism growth. Third,the use of microvolumes in microcompartments allows for the separationof a liquid test sample into a relatively larger number of test volumes.In general, the use of microvolumes in microcompartments provides a fargreater number of runs, or repetitions, of a test on the liquid sample.In the case of MPN analysis, use of microvolumes in microcompartmentsprovides a greater number of data points from which the MPN can becalculated, thereby significantly narrowing the 95% confidence limitsfor a given MPN result. Fourth, separation of sample into a large numberof test volumes allows a higher concentration of microorganisms to beenumerated, thereby reducing or eliminating sample dilutions. Fifth,this invention allows MPN analysis to be conducted in a single devicehaving the indicators and/or nutrients directly coated thereon. Sixth,this invention permits a wide counting range when performing MPNanalysis.

The foregoing has outlined some of the most pertinent objects of theinvention. These objects should be construed to be merely illustrativeof some of the more prominent features and applications of the intendedinvention. The invention includes other features and advantages whichwill be described or will become apparent from the following moredetailed description of the embodiment.

The terms “comprises” and variations thereof do not have a limitingmeaning where these terms appear in the description and claims.

The term “and/or” means one or all of the listed elements or acombination of any two or more of the listed elements.

Also herein, the recitations of numerical ranges by endpoints includeall numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.80, 4, 5, etc.).

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The description that follows more particularly exemplifiesillustrative embodiments. In several places throughout the application,guidance is provided through lists of examples, which examples can beused in various combinations. In each instance, the recited list servesonly as a representative group and should not be interpreted as anexclusive list.

Additional details of these and other embodiments are set forth in theaccompanying drawings and the description below. Other features, objectsand advantages will become apparent from the description and drawings,and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

The foregoing summary, as well as the following detailed description ofthe invention will be better understood when read in conjunction withthe appended drawings. For the purpose of assisting in the explanationof the invention, there are shown in the drawings embodiments which arepresently preferred and considered illustrative. It should beunderstood, however, that the invention is not limited to the precisearrangements and instrumentalities shown therein. In the drawings:

FIG. 1a is a plan view, partially in section, of one embodiment of thecomponents of a device for detecting microorganisms according to thepresent disclosure.

FIG. 1b is a perspective view of an assembly comprising the componentsof FIG. 1a , the assembly having the cover film in an open position forinoculating the assembly.

FIG. 1c is a cross-sectional side view of the assembly of FIG. 1B, theassembly having the cover film in a closed position.

FIGS. 2a-d are various views showing one embodiment of the use of thecomponents of FIG. 1a to partition a liquid sample and to produce adevice comprising a spacer element according to the present disclosure.

FIG. 2e is a cross sectional side view of one embodiment of thecomponents of a device for detecting microorganisms, the devicecomprising a base with a compliant member.

FIGS. 3a-3d are plan views showing one embodiment of the use of asubstrate and a cover film to partition a liquid sample and to produce adevice without a spacer element according to the present disclosure.

FIGS. 4a-4e are plan views showing one embodiment of the construction ofa pouch for analyzing a sample according to the present disclosure.

FIGS. 5a-5d are plan views of one embodiment of a method of using thepouch of FIG. 4e to partition a liquid sample.

FIG. 6 is a schematic plan view of one embodiment of the use of thedevice of FIG. 2d to detect a microorganism.

DETAILED DESCRIPTION

The present invention will now be described more fully herein after. Forthe purposes of the following detailed description, it is to beunderstood that the invention may assume various alternative variationsand step sequences, except where expressly specified to the contrary.Thus, before describing the present invention in detail, it is to beunderstood that this invention is not limited to particularlyexemplified systems or embodiments that may of course, vary. The use ofexamples anywhere in this specification including examples of any termsdiscussed herein is illustrative only, and in no way limits the scopeand meaning of the invention or of any exemplified term. Likewise, theinvention is not limited to various embodiments given in thisspecification.

Before any embodiments of the present disclosure are explained indetail, it is to be understood that the invention is not limited in itsapplication to the details of construction and the arrangement ofcomponents set forth in the following description or illustrated in thefollowing drawings. The invention is capable of other embodiments and ofbeing practiced or of being carried out in various ways. Also, it is tobe understood that the phraseology and terminology used herein is forthe purpose of description and should not be regarded as limiting. Theuse of “including,” “comprising,” or “having” and variations thereofherein is meant to encompass the items listed thereafter and equivalentsthereof as well as additional items. Unless specified or limitedotherwise, the terms “connected” and “coupled” and variations thereofare used broadly and encompass both direct and indirect connections andcouplings. Further, “connected” and “coupled” are not restricted tophysical or mechanical connections or couplings. It is to be understoodthat other embodiments may be utilized and structural or logical changesmay be made without departing from the scope of the present disclosure.Furthermore, terms such as “front,” “rear,” “top,” “bottom,” and thelike are only used to describe elements as they relate to one another,but are in no way meant to recite specific orientations of the device,to indicate or imply necessary or required orientations of the device,or to specify how the invention described herein will be used, mounted,displayed, or positioned in use.

As used herein, the singular forms “a,” “an,” and “the” include pluralreference unless the context clearly dictates otherwise. The term“and/or” means one or all of the listed elements or a combination of anytwo or more of the listed elements.

The terms “preferred” and “preferably” refer to embodiments of theinvention that may afford certain benefits, under certain circumstances.However, other embodiments may also be preferred, under the same orother circumstances. Furthermore, the recitation of one or morepreferred embodiments does not imply that other embodiments are notuseful, and is not intended to exclude other embodiments from the scopeof the invention.

When the term “about” is used in describing a value or an endpoint of arange, the disclosure should be understood to include both the specificvalue and end-point referred to.

As used herein the terms “comprises”, “comprising”, “includes”,“including”, “containing”, “characterized by”, “having” or any othervariation thereof, are intended to cover a non-exclusive inclusion.

The term “planar” as used herein refers to a plane involving twodimensions.

The expression “substantially planar” as used herein refers to a twodimensional surface. The material of the “substantially planarsubstrate” of the present invention (before forming the compartments)does not have three dimensional structure that define the ultimateshape, size, or volume of the claimed compartments.

The term “pressure sensitive adhesive” as used herein refers to anadhesive which upon application of pressure results in an adhesion withthe adherend. No solvent, water, or heat is required to bring aboutadhesion. As the name “pressure-sensitive” indicates, the degree of bondis influenced by the amount of pressure applied.

The term “hydrophobicity” as used herein refers to the physical propertyof a molecule possessing relatively little or no affinity for water oraqueous media. Hydrophobic molecules tend to be non-polar and, thus,prefer other neutral molecules and non-polar solvents. Hydrophobicmolecules in water often cluster together, forming micelles. Water onhydrophobic surfaces will exhibit a high contact angle.

The term “elastomer” as used herein refers to any polymer withviscoelasticity (having both viscosity and elasticity) and very weakinter-molecular forces, generally having low Young's modulus and highfailure strain compared with other materials. Each of the monomers whichlink to form the polymer is usually made of carbon, hydrogen, oxygenand/or silicon. Elastomers are amorphous polymers existing above theirglass transition temperature, so that considerable segmental motion ispossible. The term “elastic recovery” as used herein refers to theproportion of recovery after deformation and is quantified as percentrecovery after elongation. The elastic recovery is measured by thepercentage to which a polymer will recover its original length afterinitial elastic deformation. The higher the percentage of elasticrecovery, the greater is the tendency of the polymer to recover to itsoriginal dimensions after initial deformation.

The term “closed compartment” as used herein refers to a defined anddistinct space which retains the contents of each compartment inisolation without getting mixed with the contents of the adjacentcompartment. The expression “plurality of closed compartments” as usedherein refers to more than one such closed compartment being disposedbetween the substrate and cover film of the device as per the presentinvention.

The term “seal” as used herein refers to junction between the polymericcover film and the substrate. This junction forms the compartments andprevents leakage, prevents mixing between compartments (e.g., adjacentcompartments), and/or prevents contamination from the externalenvironment.

The term “tackifier” as used herein refers to chemical compounds used informulating adhesives to increase the tack, the stickiness of thesurface of the adhesive. They are usually low-molecular weight compoundswith high glass transition temperature. At low strain rate, they providehigher stress compliance, and become stiffer at higher strain rates.

The term “melt index” as used herein is a common measurement used tocharacterize thermoplastic polymers. It is an indirect, and inverselyproportional, measure of the viscosity of the polymer when molten. Onemeasures the mass of polymer melt which will flow through an orifice ina given amount of time under defined conditions of temperature,pressure, and geometry. The larger the melt index value, the lower isits viscosity, and therefore, the average molecular weight of thepolymer is lower. Higher molecular weight polymers will be more viscousand less will flow under the same conditions so the melt index will be asmaller number. The melt index is typically expressed in terms of gramsof polymer which flow out in a ten minute period, thus g/10 min ordg/min.

The term “specific gravity” as used herein refers to the ratio of thedensity of a substance to the density (mass of the same unit volume) ofa reference substance.

The term “optical transmittance” or “clarity” refers to the opticaldistinctness with which an object can be seen when viewed throughplastic film, sheet, glass, etc. Clarity depends upon the linearity ofthe passage of light rays through the material and is determined bysmall-angle scattering. It is the regular transmittance of a materialwhich is measured by a photoelectric detector by determining the percentof light transmitted through the material.

The term ‘liquid sample’ as used herein refers to any sample in theliquid state or may be dissolved in a liquid to form the liquefiedsample or a sample which has been liquefied.

The term “sample” may be any biological sample or environmental samplesuch as waste water, food, a surface swab, or swabs from other surfaces,such as a throat, or other samples well known to those in the art. Thissample may be a liquid sample, or may be dissolved in a liquid to formthe liquefied sample. As noted above, the biological material that canbe detected is any material that forms a discrete particle, such as amicroorganism, which may be quantified by determining the presence orabsence of such a biological material within each well of the incubationplate.

The term “anaerobic microorganism” or “anaerobe” as used herein refersto microorganisms which are sensitive to oxygen and will not grow in thepresence of oxygen. An anaerobic microorganism or anaerobe is anyorganism that does not require oxygen for growth. Anaerobicmicroorganisms include both obligate anaerobes and facultativeanaerobes. Obligate anaerobes are those microorganisms which will diewhen exposed to atmospheric levels of oxygen. A facultative anaerobe isan organism that can carry out aerobic respiration if oxygen is present,but is capable of switching to fermentation or anaerobic respiration ifoxygen is absent. Methods and systems of the present invention could beused for the enrichment and detection of both obligate anaerobes andfacultative anaerobes.

The term “culture” or “growth” of microorganisms as used herein refersto the method of increasing the number of microbial organisms by lettingthem reproduce in predetermined culture media under conditions conducivefor their growth. More particularly it is the method of providing asuitable culture medium and conditions to facilitate at least one celldivision of a microorganism. Culture media are solid, semisolid orliquid media containing all of the nutrients are required for physicalgrowth parameters essential for microbial growth.

Assemblies, devices, and methods of the present disclosure can be usedto parturition a liquid sample and, optionally, analyze the sample forthe present or absence of biomolecules and/or microorganisms. Theassemblies, devices, and methods are particularly useful in MostProbable Number (MPN) methods of analyzing a sample for the presence andquantity of microorganisms.

The most probable number method is described in InternationalPublication No. WO95/23026, which is incorporated herein by reference inits entirety. In this method, a volume of water sample is dispensed intoseveral tubes (e.g., 10×10; 10 tubes each containing 10 ml) and bacteriain each tube allowed to grow. After incubation at a specific temperaturefor a specific time, the number of positive tubes is counted. The mostprobable number in these embodiments can be determined from Formula I:

MPN/100 ml=(P×100)/(NT)^(1/2)  (Formula I)

where P is the number of positive tubes, N is the volume (ml) of samplein negative tubes, T is the volume (ml) sample in all tubes, and MPN isthe most probable number. A major drawback of the method is the range of95% confidence limits is large, when only a few tubes are used. Suchconfidence limits are calculated roughly using Formula II:

Log(MPN)±1.96(0.58/n ^(1/2))  (Formula II)

where n is the number of tests.

All of the current devices/methods in the art suffer from the problem ofhow to efficiently fill tiny compartments in a simple manner. Currentsolutions include using devices with pre-formed compartments, applying avacuum, centrifugation, and even the formation of micro drops in an oilemulsion. In each case, the method required to partition the liquid iscumbersome and real-world samples containing particulates such as foodare incompatible. Because the conventionally known methods areinadequate for routine use where rapid and easy processing of numerousand varied samples, there is need for an improved method and device.

The present invention solves the problems associated with currently usedsystems for partitioning small volumes of liquid sample into a largenumber of smaller discrete volumes. In general, the present inventionprovides a device and method to effect rapid and accurate detection andenumeration of microorganisms based on the surprising result that theuse of microvolumes substantially increases the speed of detection.Microorganisms are detected and/or enumerated not only when whole cellsare detected directly, but also when such cells are detected indirectly,such as through detection or quantization of cell fragments, cellcomponents, cell-derived biological molecules, or cell by-products.

Automated detection and enumeration of colonies on both standard agarplates, as well as PETRIFILM™ plates, is difficult and suffers from anumber of limitations. The position of each well in the stamped plateshowever is fixed, and would not require an identification algorithm. Anautomated detection platform would simply need to measure thefluorescence in a given area and determine if it is above a setthreshold. Customers that do not require the fastest possibletime-to-result would be able to observe (and enumerate) positivecompartments using ambient light or a simple hand-held illuminationsource (e.g., black light), thereby obviating the need for anauto-reader. When a sample is partitioned into many smaller samples theeffective concentration of a microorganism or other biomolecules likeviruses, nucleotides, metabolites etc., that ends up in any givenpartition is increased by orders of magnitude, increasing the speed ofreaction chemistries and reducing the time to detection.

The device of the present invention has another significant advantageover the known solid culture techniques. Advantageously, the method ofthe present invention is less labor-intensive, allows betterdistribution of sample, and provides a more accurate estimate ofmicrobial concentration. This is because the correspondingly largernumber of sample aliquots in the compartments provides a correspondinglynarrower confidence limit interval.

The present invention has manifold applications in various fields thatrequire qualitative and quantitative analysis of micro volumes of liquidsamples. The field of applications may include without limitationmicrobiology, molecular biology, biotechnology, chemistry and the like.

Exemplary microbiological applications of the qualitative andquantitative analyses include without limitation, growth assessment,monitoring, single cell enrichment cultures, isolation, performance ofsecondary tests that are not compatible with growth, most probablenumber (MPN) style testing using compartments of different sizes.

Non-limiting exemplary molecular biological applications includepolymerase chain reaction assays, detection and quantification ofbiomolecules like nucleotides, amino acids, peptides, proteins,metabolites and the like.

With reference to the Figures, a device of the present invention isconstructed as described in detail below.

As shown in FIGS. 1a-1c , a device of the present disclosure can be madefrom components that include a base 10 and a cover film 20. The basecomprises a substrate 12. In any embodiment, the substrate 12 having afirst surface 13 that is substantially planar. In any embodiment, thesubstrate 12 can be thin (e.g., less than 5 mm thick) and substantiallyflat (i.e., sheet-like). In any embodiment, the substrate 12 may beself-supporting. Optionally, the substrate 12 may be opticallytranslucent or transparent. Preferably, the substrate 12 is waterinsoluble or has a water-insoluble coating (not shown) on at least aportion of the first surface 13. Preferably, the substrate 12 will notleach any chemicals (e.g., chemicals that may inhibit microorganismgrowth and/or activity (e.g., an enzyme activity used to detect themicroorganism)) upon contact with an aqueous liquid (e.g., an aqueoussample).

In any embodiment, the base 10 and cover film 20 can be assembled intoan assembly 100 as shown in FIG. 1b . The cover film 20 may be attachedto the substrate 10 and/or spacer element 30 via a suitable attachmentmeans (e.g., a pressure-sensitive adhesive, not shown). Advantageously,a liquid sample (not shown) can be deposited into the assembly 100simply by lifting the cover film 20 to expose the area (e.g., defined byspacer element 30 surrounding the adhesive 15 and secondary coating 18(described below) of the illustrated embodiment) onto which the sampleis to be deposited.

The substrate 12 can be fabricated, for example, from polymeric films orother appropriate materials. Appropriate polymers include withoutlimitation polyethylene, polypropylene, polyester, polyimides,fluoropolymers, polycarbonates, polyurethanes, polystyrenes, derivativesthereof, and combinations thereof. Other appropriate materials mayinclude without limitation metal foils like aluminum foils, copperfoils, steel foils, laminated foils, paper foils, and paper boards. Inany embodiment, the substrate 12 is a biaxially oriented polypropylene.

The substrate 12 has a thickness of at least about 0.01 mm. In anyembodiment, the substrate 12 has a thickness greater than 5 mm, lessthan or equal to 5 mm, less than or equal to about 2 mm, less than orequal to about 1 mm. Preferably, the substrate 12 does not exhibitsubstantial light-absorbing properties (e.g., in the u.v. and/or visiblewavelengths) that would interfere with any fluorescent or color-basedindicator system that may be employed for the purposes of detection.

In any embodiment, the substrate may be optically transmissible withrespect to visible wavelengths, ultraviolet wavelengths, and/or infraredwavelengths of electromagnetic radiation.

Referring back to FIGS. 1a -lc, at least a portion of the first majorsurface 13 has a water-insoluble pressure sensitive adhesive (PSA) layer15 adhered thereto. When a device of the present disclosure is assembledas described herein, PSA layer 15 forms a bond with a cover film 20 whenpressure is applied to the cover film 20 to bring it into contact withthe PSA layer 15. No solvent, water, or heat is needed to activate thePSA adhesive and the degree of bond is influenced by the amount ofpressure (and topology of the external means, described below) which isused to contact the adhesive 15 to the cover film 20. The PSAs of thepresent invention are able to retain their adhesive properties even inthe presence of aqueous liquids and certain non-aqueous liquids that donot substantially interfere with the adhesive. The thickness of the PSAlayer coated onto the substrate 12 is at least about 0.02 mm. In anyembodiment in the thickness of the PSA layer is in the range of about0.02 mm to about 0.1 mm. Suitable PSAs include without limitationsilicone polyurea adhesive and the like.

Suitable PSAs comprise an elastomer compounded with a suitabletackifier. The pressure-sensitive adhesive is substantially insoluble inaqueous liquids (e.g., water, aqueous culture media, aqueous buffers).Neither the elastomeric compound nor the tackifier should causesubstantial inhibition of microorganism growth and/or activity. In anyembodiment, the pressure-sensitive adhesive may be opticallytransmissible with respect to visible wavelengths, ultravioletwavelengths, and/or infrared wavelengths of electromagnetic radiation.

Other suitable compositions may be based on the family ofsilicone-polyurea based pressure sensitive adhesives. Such compositionsare described in the following documents, each of which is incorporatedherein by reference in its entirety: U.S. Pat. No. 5,461,134 (Leir etal.); U.S. Pat. No. 6,007,914 (Joseph et al.); International PublicationNo. WO96/35458 (and its related U.S. patent application Ser. No.08/427,788 (filed Apr. 25, 1995); Ser. No. 08/428,934 (filed Apr. 25,1995); Ser. No. 08/588,157 (filed Jan. 17, 1996); and Ser. No.08/588,159 (filed Jan. 17, 1996)); International Publication No. WO96/34028 (and its related U.S. patent application Ser. No. 08/428,299(filed Apr. 25, 1995); Ser. No. 08/428,936 (filed Apr. 25, 1995); Ser.No. 08/569,909 (filed Dec. 8, 1995); and Ser. No. 08/569,877 (filed Dec.8, 1995)); and International Publication No. WO 96/34029 (and itsrelated U.S. patent application Ser. No. 08/428,735 (filed Apr. 25,1995) and Ser. No. 08/591,205 (filed Jan. 17, 1996)).

In any embodiment, the base 10 optionally comprises a spacer member 30attached to the substrate 10. Optionally, the spacer member 30 may beadhered to the adhesive layer 15. The spacer member 30 comprises anaperture 32 that exposes a portion of PSA layer 15 that defines theperimeter of an area used to create compartments in a device of thepresent disclosure, as described hereinbelow.

Suitable materials for the spacer member 30 include, for example, anynatural or synthetic substance which may be readily available in sheetform. Preferably, the spacer member 30 does not substantially inhibitmicroorganism growth or activity and does not absorb aqueous liquid(e.g., is constructed from or coated with hydrophobic materials).Polyethylene, polypropylene, polyethylene terephthalate and polystyreneare a few examples of suitable synthetic materials. In particular,relatively inexpensive commercially available polystyrene foams andpolyethylene foams are preferred. Natural substances such as metal e.g.foil sheets, wood and the like, optionally coated with a hydrophobiccoating, are less preferred alternatives.

The thickness of the spacer member 30 should be sufficient to create aninterior volume in the device that is large enough to hold the desiredsample volume. In any embodiment, the spacer member 30 can be less than1 mm thick, at least about 0.02 mm thick, at least about 1 mm thick, atleast about 1.5 mm thick, or at least about 2 mm thick.

In any embodiment of the present invention, the substrate coated with aprimary coating of PSA (i.e., PSA layer 15) may further comprise asecondary coating. The optional secondary coating 18 may include withoutlimitations one or more water-soluble reagents such as nutrients,dehydrated or powdered culture medium, selective agents (e.g.,antibiotics, salts) chemicals, dyes, proteins, peptides, nucleotides,enzymes and antibodies, for example. Thus, when this type of secondarycoating 18 is exposed to an aqueous liquid, the water-soluble reagentdissolves, thereby exposing the adhesive and permitting it to bond withthe cover film 20 to form compartments as described herein.

Alternatively or additionally, the secondary coating 18 may comprisewater-insoluble particles (e.g., hollow or solid glass microspheres, orfragments thereof) having a diameter that less than or equal to thethickness of the adhesive 15. With this type of secondary coating 18,the water-insoluble particles can be pushed, with or without breakage ofthe microspheres) into the adhesive layer 15 (e.g., by pressure appliedthrough the cover film, thereby exposing the adhesive and causing thecover film to bond to the adhesive, as described hereinbelow. In anyembodiment, pushing the microspheres into the adhesive layer may causebreakage of the microspheres. Advantageously, in any embodiment, thesecondary coating 18 temporarily prevents adhesion of the cover film 20to the adhesive layer 15 until a liquid is deposited into the assembly100 thereby dissolving the secondary coating (if the secondary coating18 comprises a water soluble reagent) and exposing the adhesive layer oruntil pressure is applied to the secondary coating (if the secondarycoating 18 comprises water-insoluble particles) to expose the adhesiveand thereby permit the adhesive to bond with the cover film 20.

In any embodiment, a secondary coating 18 of the present disclosure canbe substantially water-free. As used in the specification and claims,the phrase “substantially water-free” designates a coating which has awater content no greater than about the water content of the dehydratedcoating once it has been permitted to equilibrate with the ambientenvironment.

With reference to FIGS. 1a -lc, the cover film 20 can be fabricated fromany elastic polymeric film material that is non-water absorbent (e.g.,hydrophobic) and having an elastic recovery of no more than 20%. Elasticrecovery of a plastic film can be measured, for example, using ASTMD5459-95 (2012) “Standard Test Method for Machine Direction ElasticRecovery and Permanent Deformation and Stress Retention of Stretch WrapFilm”; ASTM International, West Conshohocken, Pa.; which is incorporatedherein by reference in its entirety. The cover film 20 is preferably aself-sealing, moldable and flexible film such as, for example, thecomposite film available from Bemis Flexible Packaging Company (Oshkosh,Wis.) under the trade name PARAFILM®.

In any embodiment, the cover film may be optically transmissible withrespect to visible wavelengths, ultraviolet wavelengths, and/or infraredwavelengths of electromagnetic radiation.

The cover film 20 can be fabricated, for example, from polymeric filmsor other appropriate materials. Suitable polymeric films are disclosedin U.S. Pat. No. 4,425,268, which is incorporated herein by reference inits entirety. The polymeric films may be appropriate composite ofpolymers and tackifiers. The appropriate polymers may include withoutlimitation ethylene vinyl acetate copolymer, linear copolymer ofethylene and a higher alkene. The appropriate tackifier may includewithout limitation a low molecular weight polyisobutene, polyterpenes,amorphous polypropylene, and microcrystalline wax. Advantageously coverfilm 20 is fabricated using a composition of high molecular weightcopolymer of ethylene and vinyl acetate and a linear copolymer ofethylene and a higher alkene.

The melt index of the high molecular weight copolymer is between about0.1 to about 4.0. The specific gravity of the linear copolymer isbetween about 0.917 and about 0.945. In any embodiment, the cover film20 has a thickness in the range of 0.02 mm to 0.5 mm, preferably in therange of 0.1 to 0.25 mm, more preferably in the range of 0.1 to 0.15 mm.Upon stretching of this cover film 20, the thickness of the layer maydecrease to about 0.01 mm to 0.25 mm. This stretching of the cover film20 resulting in decreased thickness of the film can contribute toefficient gaseous exchange between the sealed compartments and theirexternal environment but does not allow moisture to pass through. Thecover film 20 has low water permeability and is insensitive to moisturevapor. Advantageously, these properties of the cover film 20 and thedevice in general help in efficient culturing of aerobic and anaerobicmicroorganisms as illustrated in an embodiment of the present invention.

Returning to the drawings, FIGS. 2a-2d show one embodiment of a methodof forming a detection device (e.g., detection device 1000 of FIG. 2d )according to the present disclosure. The detection device 1000 is formedfrom an assembly 100 (FIG. 2b ) as described herein. With the cover film20 at least partially separated from the base 10 (as shown in FIG. 2a ),an aqueous sample 40 is applied (e.g., by pipet) to the base 10 of theassembly. The sample 40 is applied to the secondary coating 18, ifpresent, or the pressure sensitive adhesive 15 within an area defined bythe aperture 32 of the spacer member 30. The spacer member 30 functionsto contain the sample 40 and prevent it from escaping or leaching out.Appropriate spacer members 30 for defining the sample area may includewithout limitation bumpers, spacers, rods and metallic rims. The liquidsample 40 is dispensed in the sample area and covered with a cover film20 by bringing the cover film 20 into contact with the sample 40 and thebase 10. A person having ordinary skill in the art will recognize adevice of the present disclosure can be dimensioned to accommodatesamples having various volumes. For example, the sample volume may be assmall as about 50 μL or as large as about 100 mL or more. Preferably,the entire sample volume is distributed into the compartments of adevice according to the present disclosure. If the secondary coating 18is a water-soluble reagent, as disclosed herein, contact between theaqueous sample 40 and the secondary coating 18 will dissolve thecoating, thereby placing the adhesive layer 15 in fluid communicationwith the sample 40, as shown in FIG. 2 b.

As shown in FIG. 2b , urging the cover film 20 toward the base 10 (asshown by arrow “A” in FIG. 2a ) spreads the sample 40 so as to fill theavailable volume in the base 10 defined by the substrate 12 and thespacer member 30. Placement of the cover film 20 against the base alsoexpels substantially all of the air from the assembly 100. Efficientspreading of the liquid sample may be done in any number of ways usingmethods known in the art such as “rolling” the cover film (e.g., fromone edge of the assembly 100 to the opposite edge) onto the spacermember 30; using spreading tools like rollers, metallic spreaders,polymeric spreaders, bent glass rods, or the like; tipping the device;or applying manual pressure to the cover film, for example.

After assembling the base 10 and the cover film 20 to form the assembly100 with the liquid sample 40 disposed therein, a plurality ofcompartments are formed to produce the detection device 1000 (FIG. 2d ).In any embodiment, the compartments have a predetermined volume. In anyembodiment, the volume of each compartment of the plurality ofcompartments is about equivalent to the volume in each of the othercompartments. The sealing of the plurality of compartments at the pointof contact between the cover film 10 and the pressure sensitive adhesive15 coated on the substrate 12 is achieved by the application of pressureby external means to create a predefined pattern of seals where thecover film 20 contacts the adhesive layer 15 of the base 10. Thehydrophobicity of the cover film 20 and/or the PSA layer 15 of the base10 assists in preventing cross-contamination of the liquid confined inadjacent compartments.

The external means is employed for urging the cover film 20 to come incontact with the pressure sensitive adhesive 15 of the base 10 atspecified areas. This is achieved using any tool or device (e.g., tool50 of FIG. 2c ) with the desired patterned surface (i.e., a surfacehaving a predefined pattern of cavities) depending on the size, shapeand number of compartments desired to be achieved. The tool 50 may beany patterned device (e.g., a 96-well or 384-well microtiter plate)possessing a plurality of predetermined ridges and cavities that can beused to form complementary-patterned structures on a moldable surfacewhen the tool 50 is impressed thereupon. The size, shape, and thereforevolume of the compartments formed in the process are dictated at leastin part by the stamping tool 50 and can vary from nanoliters tomilliliters. The resulting device (e.g., device 1000 of FIG. 2d ) mayinclude any desired number of compartments 60.

In an embodiment, the tool 50 with the ridges 54 and cavities 56 ispressed against the cover film 20 of the assembly 100 (as indicated byarrow B in FIG. 2c ) leaving a pattern of wells (compartments 60 of FIG.2d ), each compartment 60 holding a given (e.g., predefined) fraction ofthe original sample (i.e., sample 40 of the device 100 shown partiallyin section in FIG. 2c ), as shown in FIG. 2d . In addition, eachcompartment 60 is surrounded by a seal 62 that fluidically isolates theportion of the sample contained in the compartment 60 from the portionsof the sample contained in the other compartments 60.

In the illustrated embodiment of FIG. 2c , the tool 50 comprises aperimeter ridge 52 that defines an area that is similar in size andshape to the aperture 32 of the spacer element 50. Advantageously, theperipheral ridge seals the perimeter of the device 1000, therebypreventing leakage of sample material from the device 1000.

The external means (e.g., tool 50) may be urged onto the cover film byany manner including without limitations press tools comprising ofhydraulic, pneumatic, mechanical and electrical types. In anyembodiment, the external means may be urged against the cover filmmanually.

A relatively large number of compartments 60 are fabricated on a singledevice 1000 using the method of the present invention. Preferably, thedevice 1000 comprises 2 to 2000 compartments, more preferably about 10to about 1000 compartments, even more preferably between about 50 toabout 500 compartments, and most preferably about 100 to about 300compartments. The device 1000 can have a population of uniformly sizedcompartments as shown in FIG. 2 d.

In any embodiment, one or more of the plurality of compartments in adevice of the present disclosure can have a volume of about 200 nL toabout 10 mL. For example, one or more of the plurality of compartmentscan have a volume of about 200 nL, about 500 nL, about 1 μL, about 10μL, about 50 μL, about 100 μL, about 250 μL, about 500 μL, about 1 mL,about 2 mL, about 5 mL, or about 10 mL.

In an embodiment, the device could include a plurality of lanes or othergroupings, each containing compartments of a particular volume, i.e.,they are not uniform throughout the device. For example, a tool asdepicted in FIG. 2 of U.S. Pat. No. 6,696,286 (which is incorporatedherein by reference in its entirety) can be used to form a device of thepresent disclosure. The corresponding device would have sets (e.g.,rows) of compartments in which volumes are constant within a set, butvary between sets. The volumes can vary incrementally over an array ofsets of compartments, with the smaller compartments holdingsub-microliter volumes, for example, and the larger compartments holdingmultiple-microliter volumes. It is even possible for the largestcompartments in a device to include compartments that would hold, forexample, up to milliliter volumes. This feature allows for thedistribution of the liquid test sample into different test volume sizeswithin a single device. For enumeration assays like most probable number(MPN), this feature would be beneficial and advantageous in that, for ahighly concentrated sample, an appropriate volume size may be selectedand MPN analysis performed using a single partitioning step in a singledevice without the need for serial dilutions.

The device of the present invention allows for discrete separation of aliquid test sample into a relatively large number of test microvolumes.The ability to separate a liquid sample into compartments and to performquantitative and qualitative analysis without cross-contaminationbetween compartments is a major advantage of the present device. Variousadditional fabrication methods, however, can be used to further enhancethe utility of the compartments, as described below.

In any embodiment of a method according to the present disclosure, thebase of a device of the present disclosure can be placed onto acompliant (i.e., flexible, yielding) surface while the external means isurged against the cover film. Advantageously, the compliant surfacefacilitates substantially uniform contact (and substantially uniformdistribution of force) between the various contact points/surfaces ofthe external means and the surface of the cover film. This uniformcontact ensures that the plurality of compartments is sealed in theprocess. Suitable compliant surfaces include, but are not limited to, alayer of flexible polymer (e.g., rubber), a layer of closed-cell oropen-cell foam (e.g., polyurethane) or the like, and combinationsthereof.

Alternatively or additionally, in any embodiment, the base of a deviceof the present disclosure further comprises a compliant member. Thecompliant member may comprise, without limitation, the aforementionedflexible polymer layers, foam layers, and combinations thereof. FIG. 2eshows one embodiment of a base 11 that comprises a compliant member 14coupled to the base 12. The compliant member 14 may be coupled to thesubstrate via any suitable means (not shown) including, for example, anadhesive, a staple, a clamp, a rivet, and a melt bond.

In any embodiment, a device of the present disclosure is formed via amethod of partitioning a liquid sample. The device need not include aspacer element. The steps of one embodiment of a method of partitioningthe aqueous sample into compartments in a device that does not have aspacer element are illustrated in FIGS. 3a-3d . A base 10 comprising asubstrate 12 having a first major surface 13 is coated with apressure-sensitive adhesive layer 15, as described herein. The base 10can be placed on a surface (preferably, a flat, substantially levelsurface) with the adhesive layer 15 facing upward. A predefined volumeof aqueous solution (sample 40 of FIG. 3a ) to be partitioned isdispensed directly onto the adhesive layer 15, as shown in FIG. 3 a.

After depositing the aqueous sample 40 onto the substrate, a cover film20 as described herein is placed onto the sample 40 and base 10 suchthat the sample 40 is disposed between the cover film 20 and theadhesive layer 15 of the base 10. Optionally, the liquid sample 40 issealed between the cover film 20 and the adhesive layer 15 of the base10 by forming a perimeter seal surrounding the liquid sample 40 disposedbetween the cover film 20 and the adhesive layer 15 of the base 10. Theperimeter seal (e.g., perimeter seal 92 shown in FIG. 3c ) is formed byurging a predefined portion of the of the cover film 20 against theadhesive layer 15. This can be done using any suitable perimeter-formingexternal means such as, for example, a tool 55 having a ridge 56surrounding a cavity 57. The cavity 57 should define a volume that is atleast as large as, a preferably about equal to, the volume of theaqueous sample 40. Thus, when the tool 55 is urged against the coverfilm, it distributes the sample 40 over a predefined area of the base10. The ridge 56 of the tool 55, when urged against the cover film 20,causes contact between the cover film 20 and the adhesive layer 15,thereby forming a perimeter seal 90 that defines a sample-holding are(i.e., a chamber 90). In any embodiment, the chamber 90 has apredetermined volume defined by the cavity 57 of the tool 55. When thevolume of the cavity 57 is approximately equal to the volume of theliquid sample 40, air is substantially excluded during formation of theperimeter seal 92. One embodiment of a suitable tool 55 form forming theperimeter seal 92 is a plastic spreading device such as the PETRIFILMYeast and Mold spreader available from 3M Company (St. Paul, Minn.).

In an alternative embodiment (not shown), the perimeter seal 92 can beformed manually by urging a blunt object (e.g., a pencil tip, an erasertip) against the cover film to trace a perimeter seal around the liquidsample. It is contemplated that the perimeter seal 92 can take the formof any one of a variety of shapes including, without limitation, acircle, an oval, a polygon, a square, a rectangle, a hexagon, anoctagon, and an obround.

After the optional perimeter seal 92 is formed, a partition-formingexternal means (e.g., tool 50 of FIG. 3c ) is urged against the coverfilm 20 to bring predefined portions of the cover film 20 into contactwith the adhesive layer 15 to form a plurality of compartments 60. Seals62 are formed where the predefined portions of the cover film 20 contactthe adhesive layer 15. The seals substantially prevent liquidcommunication between the compartments. A non-limiting example of asuitable external means for forming compartments 62 is a plastic384-well microtiter plate (Untreated black #242764, Nalge NuncInternational; Rochester, N.Y.). The external means can be urged againstthe cover film 20, for example, by using manual pressure or by using anair press (e.g., a Model A-0019 air press available from Janesville Tooland Manufacturing, Inc.; Milton, Wis.). The amount of force used to formthe seals should be enough force to ensure sufficient contact betweenthe cover film and the adhesive layer to form the seals.

Using an external means that has approximately the same shape anddimensions as the chamber 90 preferably can ensure that each compartmentof the plurality of compartments created by the method has a predefined(optionally, substantially uniform) volume. However, as illustrated inFIGS. 3a-3d , this is not mandatory.

In another aspect, the present disclosure provides a pouch forpartitioning a liquid sample (e.g., an aqueous liquid sample. FIG. 4ashows a plan view, partially in section, of some of the components usedto make a pouch according to the present disclosure. In any embodiment,a substantially planar substrate 12 has a layer or pressure-sensitiveadhesive 15 coated on a major surface. Suitable substrates 12 andadhesives 15 are described hereinabove. Prior to applying a coating overthe adhesive 15, a sheet-like mask 70 is applied (e.g., laminated) tothe adhesive 15 on the substrate 12. The mask 70 has a peripheral edge72, a central opening 74, and a gap 76 extending from the opening 74 tothe peripheral edge 72. When placed on the substrate 12, the opening 74and gap 76 expose a portion of the adhesive 15. Although shown as havinga circular shape, it is contemplated that the opening 74 may have any ofa number of suitable shapes (e.g., circular, square, oval, oblong,rectangular, polygonal, or the like).

The mask 70 can be fabricated from a variety of materials including, forexample, sheets of paper or plastic film. Preferably, the mask 70 iscoated with a low-adhesion backsize on the side that is placed againstthe adhesive 15. The low-adhesion backsize (not shown) facilitatesremoval of the mask from the adhesive 15 without disrupting the bondbetween the adhesive 15 and the substrate 12. After the mask 70 isapplied to the adhesive 15, a secondary coating 18 (e.g., a coating of apowder material such as a reagent or particles as described hereinabove)is applied to the exposed adhesive 18. FIG. 4c shows the secondarycoating 18 adheres to the portions of the adhesive that are not coveredby the mask 70. In any embodiment, the secondary coating 18 comprises awater-soluble reagent as described herein. In any embodiment, thesecondary coating comprises a plurality of particles (e.g., glassbubbles such as K37 glass bubbles available from 3M Company; St. Paul,Minn.) having a mean particle diameter less than or equal to thethickness of the layer of the adhesive 18.

After applying the secondary coating 18, excess powder optionally can beremoved (e.g., by vibration) and the mask 70 is removed. Removing themask 70 exposes the remaining adhesive 15 on the substrate 12, as shownin FIG. 4d . To complete the preparation of a pouch 500 according to thepresent disclosure, a cover film 20 dimensioned to cover the exposedadhesive 15 is laminated (e.g., using a roller, not shown) to theadhesive, as shown in FIG. 4e . The pouch 500 comprises an interiorreservoir 502 into which a liquid sample (not shown) is introducedthrough a port 504 (i.e., opening) along an edge of the pouch 500.

A pouch 500 according to the present disclosure can be used in a methodof partitioning a sample. FIGS. 5a-5d show one embodiment of the stepsthat are used to partition a liquid sample using the pouch 500 of FIG. 4e.

A liquid sample 40 (e.g., an aqueous sample suspected of containing abiological material (e.g., a microorganism or biomolecule) is introduced(e.g., via pipet) into the reservoir 502 of the pouch 500, as shown inFIG. 5a . If the secondary coating (not shown) in the reservoir 502comprises a water-soluble reagent, introduction of the liquid sample 40dissolves the reagent and exposes the adhesive (not shown so that it canbond with the cover film of the pouch 502. Optionally, a spreadingdevice 55 (e.g., a PETRIFILM yeast and mold plate spreader availablefrom 3M Company; St. Paul, Minn.) can be urged against the cover film ofthe pouch 500 to spread the liquid throughout the reservoir 502, to pushair out of the reservoir 502 (via the port), and to form a peripheralseal 92 around a single, liquid-filled chamber 90, as shown in FIG. 5b .An external means (e.g., tool 50) is urged against the liquid-filledreservoir (not shown) or liquid-filled compartment 90 (as shown in FIG.5c ), as described above, to form the device 2000 shown in FIG. 5d . Thedevice 2000 comprises a plurality of liquid-filled compartments 40, eachcompartment isolated from adjacent compartments via one or more seal 42according to the pattern of ridges 54 defined by the tool 50 asdescribed herein.

Accordingly, the present invention provides a method for detecting amicroorganism in a test sample. Non-limiting examples of suitable testsamples include solids, semi-solids, gelatinous materials, particulatesuspensions, solutions, liquids, and combinations thereof. Solid orsemi-solid samples can be homogenized and/or suspended in an aqueousmedium (e.g., sterile water, a buffer, a nutrient medium) before theyare introduced into a device or pouch of the present disclosure. Theliquid or liquefied test sample may be deposited directly on to thesubstrate coated with adhesive and, optionally a secondary coating. Thesample is spread out and the device is formed as described herein,resulting in the partitioning of the liquid into a plurality of closedcompartments disposed between the substrate and the cover film byefficient sealing of the top and substrates with the aid of the PSA. Thequalitative and quantitative analysis of the partitioned samples can beconducted on at least one compartment using the methods known to aperson skilled in the art.

Quantitative analysis include without limitation enumeration,quantification, counting and measurement of the microorganisms orbiomolecules in the sample. Biomolecules may include without limitationpolysaccharides, lipids, nucleic acids, DNA, RNA, metabolites, vitamins,hormones and amino acids. Qualitative analysis include withoutlimitation detection, culturing, isolation, identification andpurification of microorganisms or biomolecules.

In another embodiment, the invention relates to a method for culturing amicroorganism in a liquid test sample. The method is similar to themethod described above except that the partitioned samples include anutrient growth medium and are allowed to incubate under conditions fora time sufficient to facilitate at least one cell division of themicroorganism. For the culturing of anaerobic microorganisms, the devicewith the partitioned samples may be kept in an anaerobic chamber tomaintain an anaerobic environment in the compartments.

After distribution of the sample into compartments, various assays maybe carried out depending on desired uses. For microbial detection orenumeration, the assay device may be incubated for a time sufficient topermit at least one cell division cycle of the microorganism. For thesepurposes, the device is generally incubated at about 25° C. to about 45°C., more preferably at about 30° C. to about 37° C. The incubation timefor microorganism detection will vary. The detection time will also varydepending on the growth rate, the detection system (e.g., indicatorreagents) used, and the number of microorganisms present in the sample.

The liquid test sample may be any sample of interest, from any source.The liquid test sample may include selective nutrient growth media forthe microorganism of interest, and/or an indicator substance thatproduces a signal in the presence of the growing microorganism.Preferably, nutrient growth media is present as a coating on thesubstrate, in amounts sufficient to achieve desired concentrations whena volume of the liquid test sample is distributed onto the substrate.Such a coating may be achieved, for example, by placing or distributinga solution of the nutrient media onto the substrate and drying thesolution to produce a coating or deposition of the nutrient medium onthe film. Components of the media may be present in the adhesive coatedon the substrate. The media ultimately diffuses into the sample when itcomes into contact with the liquid sample.

A wide variety of selective growth media for a wide variety ofmicroorganisms of interest is known, as is a wide variety of indicatorsubstances for a wide variety of microorganisms, and any of these mediaor indicator substances are suitable for use in the method of theinvention. An advantage of the present invention is that solubleindicators can be used, since diffusion is prevented by confinement ofthe aqueous biological sample in the sealed compartments.

In other embodiments, the compartments may contain a coating of nutrientmedium, and the nutrient medium may further include at least oneindicator substance. Alternatively, the liquid test sample may includeat least one indicator substance. In either case, the indicatorsubstance may be any indicator substance capable of providing adetectable signal in the liquid test sample. Such indicators include,but are not limited to, chromogenic indicators, fluorescent indicators,luminescent indicators, and electrochemical indicators. For purposes ofthis application, the term “electrochemical” means a chemical indicatorthat changes the resistance or conductance of the sample upon reactionwith a microorganism.

The assay reagents can be immobilized in the substrate by any ofnumerous methods for immobilizing assay reagents on solid substratesknown to those of skill in the art. Such methods include for exampledrying down assay reagent-containing liquids, and other methods fornon-covalently attaching biomolecules and other assay reagents to asolid substrate. Alternatively, various methods may be employed tocovalently attach assay reagents to the substrate by methods well knownto those of skill in the art.

Fluorogenic indicators which are detected at relatively lowconcentrations may be suitably employed. Suitable indicators include4-methylumbelliferyl phosphate, and4-methylumbelleferyl-β-D-glucopyranoside,L-phenylalanine-7-amido-4-methylcoumarin. Others may include4-methylumbelliferyl acetate and 4-methylumbelliferyl sulfate.

In another aspect, a kit for the device is fabricated. In anyembodiment, the kit comprises the following components: (i) a substratehaving a first major surface with a layer of a water-insoluble pressuresensitive adhesive coated thereon and (ii) a composite polymeric film.The substrate can be any suitable substrate for the device as describedherein. The composite polymeric film comprises ethylene vinyl acetatecopolymer, a linear copolymer of ethylene and a higher alkene, and atackifier. The tackifier is selected from a group consisting of a lowmolecular weight polyisobutene, polyterpenes, amorphous polypropylene,and microcrystalline wax. In any embodiment, the composite polymericfilm has an elastic recovery less than or equal to 20%.

In any embodiment, the kit comprises the following components: (i) asubstrate having a first major surface with a layer of a water-insolublepressure sensitive adhesive adhered thereto, the substrate and adhesiveas described herein, and (ii) a polymeric film having an elasticrecovery of less than or equal to 20%, as described herein.

In any embodiment of the kit, the substrate and/or the polymeric film issubstantially planar. In any embodiment of the kit, the substrate and/orthe polymeric film is substantially flat. In any embodiment of the kit,the pressure sensitive adhesive comprises silicone polyurea. In anyembodiment of the kit, the substrate further comprises a secondarycoating disposed on at least a portion of the adhesive. In anyembodiment of the kit, the secondary coating comprises a powderednutrient and/or a plurality of glass bubbles. In any embodiment of thekit, the secondary coating is substantially water-free. In anyembodiment of the kit, a spacer element is coupled to the first majorsurface of the substrate, as described hereinabove. In any embodiment ofthe kit, the composite polymeric film is attached to the substrate,wherein the spacer element, if present, is disposed between thesubstrate and the cover film.

Additional components of any kit according to the present disclosure maycomprise sample spreading tools, sample area defining objects andstamping tools of various sizes and shapes. The kit may additionallycomprise instruction manual for ease of use.

There are a variety of alternative techniques and procedures availableto those of skill in the art that would similarly permit one tosuccessfully practice the intended invention. All specific materials andmethods described below, in whole or in part, fall within the scope ofthe invention. These specific compositions, materials, and methods arenot intended to limit the invention, but merely to illustrate specificembodiments falling within the scope of the invention. One skilled inthe art may develop equivalent materials, and methods without theexercise of inventive capacity and without departing from the scope ofthe invention. It will be understood that many variations can be made inthe procedures herein described while still remaining within the boundsof the invention. It is the intention of the inventors that suchvariations are included within the scope of the invention.

Exemplary Embodiments

Embodiment A is a device, comprising:

a base comprising a substrate, the substrate having a first majorsurface;

a pressure sensitive adhesive adhered to at least a portion of the firstmajor surface;

a polymeric cover film coupled to the substrate via the adhesive;

-   -   wherein the cover film is a composite film comprising a polymer        and a tackifier;    -   wherein the composite film comprises ethylene vinyl acetate        copolymer, a linear copolymer of ethylene and a higher alkene,        and the tackifier;

a plurality of closed compartments disposed between the substrate andthe cover film, each compartment of the plurality defined by a seal thatprevents liquid communication with at least one other compartment of theplurality; and

an aqueous liquid disposed in two or more of the closed compartments;

wherein the seal is formed by contact between the cover film and thepressure-sensitive adhesive.

Embodiment B is the device of Embodiment A, wherein the tackifier isselected from a group consisting of a low molecular weightpolyisobutene, polyterpenes, amorphous polypropylene, andmicrocrystalline wax.

Embodiment C is the device of Embodiment A or Embodiment B, wherein thecover film has an elastic recovery less than or equal to 20%.

Embodiment D is a device, comprising:

a base comprising a substrate, the substrate having a first majorsurface;

a pressure sensitive adhesive adhered to at least a portion of the firstmajor surface;

a polymeric cover film coupled to the substrate via the adhesive, thecover film having an elastic recovery less than or equal to 20%;

a plurality of closed compartments disposed between the substrate andthe cover film, each compartment of the plurality defined by a seal thatprevents liquid communication with at least one other compartment of theplurality; and

an aqueous liquid disposed in two or more of the closed compartments;wherein the seal is formed by contact between the cover film and thepressure-sensitive adhesive.

Embodiment E is the device of any one of the preceding Embodiments,wherein the substrate is water insoluble.

Embodiment F is the device of any one of the preceding Embodiments,wherein the seal prevents liquid communication between any twocompartments of the plurality.

Embodiment G is the device of any one of the preceding Embodiments,wherein the first major surface is substantially planar.

Embodiment H is the device of any one of the preceding Embodiments,wherein the substrate has a thickness of at least about 0.02 mm.

Embodiment I is the device of Embodiment H, wherein the substrate has athickness less than or equal to 5 mm.

Embodiment J is the device of Embodiment H, wherein the substrate has athickness less than or equal to 2 mm.

Embodiment K is the device of any one of the preceding Embodiments,wherein the substrate is made of a material selected from the groupconsisting of polypropylene, polyurethane, polyethylene, polyester,polyimide, fluoropolymers, polycarbonate, polystyrene, a derivative ofany one of the foregoing materials, and a combination of any two or moreof the foregoing materials.

Embodiment L is the device of Embodiment K, wherein the material ispolypropylene, wherein the polypropylene is biaxially-orientedpolypropylene.

Embodiment M is the device of any one of the preceding Embodiments,wherein the substrate is comprises a metal foil.

Embodiment N is the device of Embodiment M, wherein the metal foilcomprises aluminum, copper, or steel.

Embodiment O is the device of Embodiment M or Embodiment N, wherein themetal foil comprises a metal foil-polymer laminate.

Embodiment P is the device of any one of the preceding Embodiments,wherein the pressure sensitive adhesive retains its adhesive propertywhen in contact with an aqueous liquid.

Embodiment Q is the device of any one of the preceding Embodiments,wherein the pressure sensitive adhesive comprises an elastomer and atackifier.

Embodiment R is the device of any one of Embodiments A through P,wherein the pressure sensitive adhesive is silicone polyurea adhesive.

Embodiment S is the device of any one of the preceding Embodiments,wherein the thickness of the pressure sensitive adhesive is at leastabout 0.02 mm.

Embodiment T is the device of Embodiment S, wherein the thickness of thepressure sensitive adhesive is less than or equal to 0.2 mm.

Embodiment U is the device of any one of the preceding Embodiments,wherein the cover film is a composite film comprising a polymer and atackifier.

Embodiment V is the device of any one of the preceding Embodiments,wherein the cover film is a self-sealing, moldable and flexible film.

Embodiment W is the device of any one of the preceding Embodiments,wherein the thickness of at least a portion of the cover film is 0.01 mmto 0.5 mm.

Embodiment X is the device of Embodiment W, wherein the thickness of atleast a portion of the cover film is 0.10 mm to 0.25 mm.

Embodiment Y is the device of any one of the preceding Embodiments,wherein the thickness of at least a portion of the cover film whenstretched is 0.01 mm to 0.20 mm.

Embodiment Z is the device of any one of the preceding Embodiments,wherein the aqueous liquid comprises a biological sample.

Embodiment AA is the device of Embodiment Z, wherein the biologicalsample is for microbiological analysis or biochemical analysis.

Embodiment AB is the device of Embodiment Z, wherein the biologicalsample is a food sample, clinical sample, an environmental sample, or awaste water sample.

Embodiment AC is the device of any one of the preceding Embodiments,wherein the aqueous liquid in each compartment has a volume of 200 nL to10 mL.

Embodiment AD is the device of any one of the preceding Embodiments,wherein the aqueous liquid in all of the compartments has a total volumein the range of 50 μL to 100 mL.

Embodiment AE is the device of any one of the preceding Embodiments,wherein the device further comprises a spacer element disposed betweenthe substrate and the cover film.

Embodiment AF is the device of Embodiment AE, wherein the spacer elementis attached to the substrate.

Embodiment AG is the device of any one of the preceding Embodiments,wherein the device further comprises a secondary coating disposed on atleast a portion of the pressure sensitive adhesive.

Embodiment AH is the device of Embodiment AG, wherein the secondarycoating consists essentially of dry powder.

Embodiment AI is the device of Embodiment AG or Embodiment AH, whereinthe secondary coating is selected from the group consisting ofnutrients, chemicals, dyes, proteins, enzymes and antibodies.

Embodiment AJ a method for partitioning liquid comprising:

depositing a predefined volume of liquid between a substrate and apolymeric cover film wherein said substrate is coated withwater-insoluble pressure sensitive adhesive;

-   -   wherein the cover film is a composite film comprising a polymer        and a tackifier;    -   wherein the composite film comprises ethylene vinyl acetate        copolymer, a linear copolymer of ethylene and a higher alkene,        and the tackifier; and

urging an external means against the cover film to bring discreteregions of the cover film in contact with the pressure sensitiveadhesive of the substrate resulting in the partitioning of the liquidinto a plurality of closed compartments disposed between the substrateand the cover film.

Embodiment AK is the method of Embodiment AJ, wherein the tackifier isselected from a group consisting of a low molecular weightpolyisobutene, polyterpenes, amorphous polypropylene, andmicrocrystalline wax.

Embodiment AL is the method of Embodiment AJ or Embodiment AK, whereinthe cover film has an elastic recovery less than or equal to 20%.

Embodiment AM is a method for partitioning liquid comprising:

depositing a predefined volume of liquid onto a first surface of asubstrate such that the liquid sample is disposed between the firstsurface and a polymeric cover film, wherein said first surface is coatedwith water-insoluble pressure sensitive adhesive and said cover film hasan elastic recovery less than or equal to 20%; and

urging an external means against the cover film to bring discreteregions of the cover film in contact with the pressure sensitiveadhesive of the substrate resulting in the partitioning of the liquidinto a plurality of closed compartments disposed between the substrateand the cover film.

Embodiment AN is the method of any one of Embodiments AJ through AMwherein, prior to urging the external means against the cover film, thesubstrate and/or the cover film are substantially flat.

Embodiment AO is the method of any one of Embodiments AJ through AN,wherein the external means comprises a patterned surface having aplurality of cavities.

Embodiment AP is the method of any one of Embodiments AJ through AO,wherein the substrate further comprises a secondary coating disposed onthe adhesive.

Embodiment AQ is the method of Embodiment AP, wherein the secondarycoating is selected from the group consisting of nutrients, chemicals,dyes, proteins, enzymes and antibodies.

Embodiment AR is a method for analyzing a liquid sample, comprising:

depositing a predefined volume of liquid onto a first surface of asubstrate such that the liquid sample is disposed between the firstsurface and a polymeric cover film wherein said first surface is coatedwith water-insoluble pressure sensitive adhesive;

-   -   wherein the cover film is a composite film comprising a polymer        and a tackifier;    -   wherein the composite film comprises ethylene vinyl acetate        copolymer, a linear copolymer of ethylene and a higher alkene,        and the tackifier;

urging an external means against the cover film to bring discreteregions of the cover film in contact with the pressure sensitiveadhesive of the substrate resulting in the partitioning of the liquidinto a plurality of closed compartments disposed between the substrateand the cover film; and

conducting a quantitative analysis or a qualitative analysis of at leastone closed compartment of the plurality.

Embodiment AS is the method of Embodiment AR, wherein the tackifier isselected from a group consisting of a low molecular weightpolyisobutene, polyterpenes, amorphous polypropylene, andmicrocrystalline wax.

Embodiment AT is the method of Embodiment AR or Embodiment AS, whereinthe cover film has an elastic recovery less than or equal to 20%.

Embodiment AU is a method for analyzing a liquid sample comprising:

depositing a liquid sample onto a first surface of a substrate such thatthe liquid sample is disposed between the first surface and a polymericcover film wherein said first surface is coated with water-insolublepressure sensitive adhesive and said polymeric cover film has an elasticrecovery less than or equal to 20%;

(ii) urging an external means against the cover film to bring discreteregions of the cover film in contact with the pressure sensitiveadhesive of the substrate resulting in the partitioning of the liquidinto a plurality of closed compartments disposed between the substrateand the cover film; and

conducting a quantitative analysis or a qualitative analysis of at leastone closed compartment of the plurality.

Embodiment AV is the method of any one of Embodiments AR through AUwherein, prior to urging the external means against the cover film, thesubstrate and/or the cover film are substantially flat.

Embodiment AW is the method of any one of Embodiments AR through AV,wherein the quantitative analysis comprises enumeration ofmicroorganisms or biomolecules in the sample.

Embodiment AX is the method of Embodiment AW, wherein the biomoleculesare selected from a group consisting of proteins, polysaccharides,lipids, nucleic acids, DNA, RNA, metabolites, vitamins, hormones andamino acids.

Embodiment AY is the method of any one of Embodiments AR through AX,wherein the qualitative analysis comprises detection, culturing,isolation, identification or purification of a microorganism or abiomolecule in the sample.

Embodiment AZ is a method for culturing a microorganism, comprising:

depositing a predefined volume of liquid onto a first surface of asubstrate such that the liquid sample is disposed between the firstsurface and a polymeric cover film wherein said first surface is coatedwith water-insoluble pressure sensitive adhesive;

-   -   wherein the cover film is a composite film comprising a polymer        and a tackifier;    -   wherein the composite film comprises ethylene vinyl acetate        copolymer, a linear copolymer of ethylene and a higher alkene,        and the tackifier;

urging an external means against the cover film to bring discreteregions of the cover film in contact with the pressure sensitiveadhesive of the substrate resulting in the partitioning of the liquidinto a plurality of closed compartments disposed between the substrateand the cover film; and

incubating the liquefied and partitioned sample, under conditions tofacilitate at least one cell division of said microorganism.

Embodiment BA is the method of Embodiment AZ, wherein the tackifier isselected from a group consisting of a low molecular weightpolyisobutene, polyterpenes, amorphous polypropylene, andmicrocrystalline wax.

Embodiment BB is the method of Embodiment AZ or Embodiment BA, whereinthe cover film has an elastic recovery less than or equal to 20%.

Embodiment BC is a method for culturing a microorganism comprising:

mixing a sample with a liquid nutrient medium to render it liquefied;

depositing the liquefied sample onto a first surface of a substrate suchthat the liquid sample is disposed between the first surface and apolymeric cover film wherein said first surface is coated withwater-insoluble pressure sensitive adhesive and said polymeric coverfilm has an elastic recovery less than or equal to 20%;

urging an external means against the cover film to bring discreteregions of the cover film in contact with the pressure sensitiveadhesive of the substrate resulting in the partitioning of the liquidinto a plurality of closed compartments disposed between the substrateand the cover film; and

incubating the liquefied and partitioned sample under conditions tofacilitate at least one cell division of said microorganism.

Embodiment BD is the method of any one of Embodiments AZ through BCwherein, prior to urging the external means against the cover film, thesubstrate and/or the cover film are substantially flat.

Embodiment BE is the method of any one of Embodiments AZ through BD,wherein the microorganism is aerobic or anaerobic.

Embodiment BF is a kit, comprising:

a substrate having a first major surface with a layer of awater-insoluble pressure sensitive adhesive coated thereon; and

a composite polymeric film;

-   -   wherein the polymeric film comprises ethylene vinyl acetate        copolymer, a linear copolymer of ethylene and a higher alkene,        and a tackifier;    -   wherein the tackifier is selected from a group consisting of a        low molecular weight polyisobutene, polyterpenes, amorphous        polypropylene, and microcrystalline wax.

Embodiment BG is the kit of Embodiment BF, wherein the compositepolymeric film has an elastic recovery of less than or equal to 20%.

Embodiment BH is a kit comprising:

a substrate having a first major surface with a layer of awater-insoluble pressure sensitive adhesive adhered thereto; and

a polymeric film having an elastic recovery of less than or equal to20%.

Embodiment BI is the kit of any one of Embodiments BF through BH,wherein the substrate and/or the polymeric film is substantially planar.

Embodiment BJ is the kit of any one of Embodiments BF through BI,wherein the pressure sensitive adhesive comprises silicone polyurea.

Embodiment BK is the kit of any one of Embodiments BF through BJ,wherein the substrate further comprises a secondary coating disposed onat least a portion of the adhesive.

Embodiment BL is the kit of Embodiment BK, wherein the secondary coatingcomprises a powdered nutrient and/or a plurality of glass bubbles.

Embodiment BM is the kit of any one of Embodiments BF through BL,wherein a spacer element is coupled to the first major surface of thesubstrate.

Embodiment BN is the kit of any one of Embodiments BF through BM:

wherein the polymeric film is attached to the substrate;

wherein the spacer element, if present, is disposed between thesubstrate and the cover film.

Embodiment BO is a pouch, comprising:

a substrate;

a composite polymeric film attached to the substrate;

a reservoir disposed between the first layer and the second layer; and

a port through which a liquid can be introduced into the reservoir;

wherein, in the reservoir, the substrate includes a pressure-sensitiveadhesive layer adhered to the substrate and a substantially water-freesecondary layer coated onto the adhesive layer;

wherein the secondary layer prevents adhesion between the adhesive andthe composite polymeric film;

wherein the polymeric film comprises ethylene vinyl acetate copolymer, alinear copolymer of ethylene and a higher alkene, and a tackifier;

wherein the tackifier is selected from a group consisting of a lowmolecular weight polyisobutene, polyterpenes, amorphous polypropylene,and microcrystalline wax.

Embodiment BP is the pouch of Embodiment BO, wherein the compositepolymeric film has an elastic recovery of less than or equal to 20%.

Embodiment BQ is a pouch, comprising:

a substrate;

a polymeric film attached to the substrate;

a reservoir disposed between the substrate and the polymeric film; and

a port through which a liquid can be introduced into the reservoir;

wherein, in the reservoir, the substrate includes a pressure-sensitiveadhesive layer adhered to the substrate and a substantially water-freesecondary layer coated onto the adhesive layer;

wherein the secondary layer prevents adhesion between the adhesive andthe composite polymeric film;

wherein the polymeric film has an elastic recovery of less than or equalto 20%.

Embodiment BR is the pouch of any one of Embodiments BO through BQ,wherein the secondary coating comprises a powder.

Embodiment BS is the pouch of any one of Embodiments BO through BR,wherein the secondary coating comprises a nutrient, a reagent forindicating microbial growth, or a selective agent.

Embodiment BT is the pouch of any one of Embodiments BN through BS,wherein the secondary coating comprises a plurality of water-insolubleparticles.

Embodiment BU is the pouch of Embodiment BT, wherein the water-insolubleparticles comprise glass bubbles.

Embodiment BV is the pouch of Embodiment BT or Embodiment BU, whereinthe water-insoluble particles have a mean diameter, wherein the adhesivelayer has a thickness, wherein the mean diameter is less than or equalto the thickness.

Embodiment BW is a kit comprising the pouch of any one of Embodiments BQthrough BX.

Examples

Objects and advantages of this invention are further illustrated by thefollowing examples, but the particular materials and amounts thereofrecited in these examples, as well as other conditions and details,should not be construed to unduly limit this invention. Unless otherwiseindicated, all parts and percentages are on a weight basis, all water isdistilled water, and all molecular weights are weight average molecularweight.

Example 1: Preparation of Sealed Device and Partitioning of AqueousSample in the Sealed Device

Preparation of the Substrate and Cover Film

Biaxially oriented polypropylene of 0.05 mm (2 mil) thickness, having asubstantially planar first surface, was used (BOPP, 2 mil) as thesubstrate. On the first surface, the film was coated with awater-insoluble, silicone based pressure sensitive adhesive, siliconepolyurea, of 0.05 mm (2 mil) thickness. The water-insoluble siliconepolyurea adhesive was prepared according to the method described in U.S.Pat. Nos. 5,461,134 and 6,007,914; which are both incorporated herein byreference in their entirety.

For the hydrophobic cover film, plastic paraffin film (PARAFILM M 4 mil(0.1 mm) film, Bemis Flexible Packaging Company; Oshkosh, Wis.) of 0.1mm thickness was used.

Partitioning of the Aqueous Sample into Compartments

Aqueous solutions were prepared by diluting an overnight culture ofEscherichia coli (ATCC #25922, American Type Tissue Collection;Manassas, Va.) in BACTO™ Trypticase Soy Broth (Becton, Dickinson andCompany; Franklin Lakes, N.J.) with 0.5 mg/ml4-methylumbelliferyl-β-D-glucuronide (Sigma-Aldrich Corp.; St. Louis,Mo.). The test solutions had about 1-100 bacteria per microliter.

The steps in the process of partitioning the aqueous sample intocompartments are illustrated in FIGS. 3a-3d . The adhesive-coatedsubstrate (i.e., the base 10 of FIG. 3a ) was placed on a flat surfacewith the adhesive 15 facing upward and 1 ml of test aqueous solution(sample 40 of FIG. 3a ) was dispensed directly onto the adhesive-coatedbase, as shown in FIG. 3 a.

After adding the aqueous sample onto the substrate, the cover film 20was then carefully placed onto the sample 40 and base 10 and a plasticspreading device (a PETRIFILM yeast and mold plate spreader, 3M Company;St. Paul, Minn.) was urged against the cover film 20 (as indicated byarrow B in FIG. 3b ) to spread the liquid out and seal the liquid into acircular chamber (chamber 90 of FIG. 3c ), having a 6 cm diameter,between the cover film 20 and the base. The chamber 90 was bordered by aperimeter seal 92 that joined the cover film 20 to the base 10.

A plastic 384-well microtiter plate (Untreated black #242764, Nalge NuncInternational; Rochester, N.Y.) (tool 50 of FIG. 3c ) was placed on topof the culture device with the openings of the wells facing down and anair press (Model A-0019, Janesville Tool and Manufacturing, Inc.;Milton, Wis.) was used to press the microtiter plate down using moderateforce (i.e., using just enough force to ensure contact between the coverfilm and the adhesive layer), deforming the cover film up into thecavities of the microtiter plate and sealing the cover film to thesubstrate in the area between the wells, thereby forming the inoculatedpartitioned device 2000 of FIG. 3 d.

The compartments 60 (FIG. 3d ) were formed by the creation of seals 62at the area of contact between the cover film and the pressure-sensitiveadhesive of the base. The resulting seals 62 formed leak proofcompartments.

After stamping, each device contained approximately 140 individuallysealed wells that each contained between 5 and 10 microliters of sample.Deformation of the cover film appeared to be very uniform with >95% ofthe wells being completely liquid-filled with no visible air bubbles.The stamped culture device was subsequently removed from the 384-wellplate.

Example 2: Compartments Having Acrylate Adhesive-Coated Aluminum Foil asBottom Layer

A culture device was constructed as described in Example 1 above, withexceptions as noted in the following paragraph.

For the substrate, 9792R foil tape (3M Company, St. Paul, Minn.) wasused. This foil (0.036 mm thick) is a dead soft aluminum foil coated onone side with a 3M select diagnostic acrylate adhesive. The adhesive isapproximately 0.027 mm thick. The foil is opaque, pierceable and theadhesive is very compatible to bioassays.

The cover film was fabricated using PARAFILM M plastic paraffin film, asdescribed in Example 1. The construction of this device permitted a 1 μlcapillary pipette to be inserted through the foil tape in order toextract sample from a any of the individual compartments (not shown).Advantageously, this can permit subsequent tests (e.g., biochemical,immunological, enzymatic tests) to be performed using at least a portionof the contents of a particular compartment.

Example 3: Detection of Bacterial Growth—Qualitative Analysis

Incubation and Detection of Growth of Microorganism in Compartments

The stamped culture device of Example 1 having the partitioned samplesin the sealed compartments was incubated at 37° C. for 24 hours.

Aerobic count (AC) PETRIFILM plates (3M Company; St. Paul, Minn.) wereused as a comparator with 1 ml of the same test solution having beeninoculated onto the film according to the manufacturer's instructions,spread, and incubated at 37° C. for 24 hours.

After the incubation, the liquid-filled compartments were observed forgrowth of bacteria as evidenced by fluorescence in one or morecompartment when the device was place under UV illumination. Enzymaticcleavage of the non-fluorescent 4-methylumbelliferyl-β-D-glucuronide(MUG) to a fluorescent product by E. coli β-glucuronidase indicatedpositive growth in a compartment. The hydrolysis of the4-methylumbelliferyl-β-D-glucuronide (MUG) fluorogenic substrate wasdetected using stereomicroscope. A stereomicroscope (Zeiss Luminar V12with Axiocam MRc5) equipped with an excitation source (365 nm) and anemission filter (400 nm long pass) was used to visualize positivecompartments. The results are shown in Table 1.

Examples shown here were chosen because it is statistically likely(p<0.05) that only a single organism was partitioned from the originalaqueous test solution into each well that yielded a positivefluorescence signal.

TABLE 1 Growth of E. coli in a culture device according to the presentdisclosure. Example 1 PETRIFILM Plates (Growth-positive^(a)) (CFU) TestSolution 1 (5 μL)  21 compartments 4 Test Solution 2 (10 μL) 130compartments 82 ^(a)Growth was evidenced by positive fluorescence andthe absence of observable gas bubbles in each “growth-positive”compartment. Fluorescence in a compartment was considered positive forgrowth of the bacterium in the compartment.

In addition to using fluorescent indicators it was noticed thatnon-fluorescent wells had accumulated small gas bubbles during theincubation period while florescent wells (positive for growth) did not.The absence of bubbles in a compartment correlated with bacterial growthin the compartment.

Example 4: Detection of Bacterial Redox Activity and Growth

Stamped culture devices were prepared according to Example 1, using 0.1or 0.01 mg/ml resazurin (Sigma-Aldrich Corp.; St. Louis, Mo.) instead of0.5 mg/ml MUG (4-methylumbelliferyl-β-D-glucuronide). Addition ofresazurin allows for detection of bacterial redox activity and does notrely on a specific enzyme activity (β-D-glucuronidase), as does MUG, todetect microbial activity.

The stamped culture devices were incubated as provided in Example 2above. Aerobic count (AC) PETRIFILM plates were used as a comparatorwith 1 ml of the same test sample.

Detection of the bacterial growth was done by visual inspection of theliquid-filled compartments. Dark pink wells considered negative forbacterial growth and light pink to clear compartments were consideredpositive for bacterial growth. The results are shown in Table 2. As inExample 2 above, the absence of small gas bubbles within a compartmentalso was an accurate indicator of bacterial growth.

TABLE 2 Example 3 PETRIFILM Plates (Growth-positive^(a)) (CFU)  0.1mg/ml Resazurin 18 compartments 4 0.01 mg/ml Resazurin 24 compartments 3^(a)Growth was evidenced by a color change and the absence of observablegas bubbles in each “growth-positive” compartment of Example 3.

Example 5: Time-to-Result for Detection of Continuous Growth of Bacteria

A culture device was constructed and inoculated as in Example 1 usingthe COLILERT® bacteriological medium (IDEXX laboratories; Westbrook,Me.) without added 4-methylumbelliferyl-β-D-glucuronide (MUG) since MUGis already contained within the COLILERT medium.

The device was positioned such that the compartments formed by the coverfilm were facing into the wells of a plastic 384-well microtiter plate(Untreated black #242764, Nalge Nunc International; Rochester, N.Y.).This assembly was subsequently placed into the reading tray of a TecanINFINITE® M200 plate reader (Tecan Systems, Inc., San Jose, Calif.) andscanned for fluorescence. In this configuration the light source anddetector were above the device resulting in excitation and emissionlight passed through the substrate of the culture device.

The plate reader chamber was set to hold a temperature of 37° C. andfluorescence measurements were taken every 20 minutes for 24 hours usingan excitation wavelength of 365 nm and an emission filter of 470±10 nm.The resulting fluorescence curves were plotted as a function of time. Acompartment of the culture device was determined to be positive forgrowth when the fluorescence at any given time point was 2.5-foldgreater than the average of the first 18 time points (6 hours) for thatcompartment. The time point (minutes of incubation at 37° C.) at whichpositive growth was observed in each compartment is reported in Table 3.

TABLE 3 Time to detection. This table reports the number of minutes ofincubation required to observe positive growth in each compartment of astamped culture device of the present disclosure. Column Number C D E FG H I J K L M N O Row 6 — — — — — 820 680 NG NG — — — — Number 7 — — —NG 720 700 740 760 660 NG — — — 8 — NG NG  760 740 760 780 800 660 NG NGNG — 9 — 740 NG NG 740 640 NG NG NG 840 780 NG — 10 NG NG 660 NG NG NGNG NG NG NG 780 NG NG 11 NG 980 NG NG NG NG 660 980 NG NG 740 760 640 12NG 700 NG NG NG NG 680 700 840 800 NG NG NG 13 720 680 780  760 800 820660 760 NG 680 NG 680 NG 14 NG NG 700  880 NG 700 NG NG NG NG 760 700640 15 NG NG NG NG 640 NG 740 NG 740 NG NG 980 — 16 — — NG NG 680 NG 780760 NG 740 NG NG — 17 — — — 1140 660 700 720 640 680 700 840 — — 18 — —— — 720 NG NG 780 700 NG — — — “NG” = no growth (i.e., the fluorescencein the compartment did not exceed the stated threshold for determiningmicrobial growth.

Rows and columns of the compartments are designated on the left side andtop respectively. Numbers in each cell of the table indicatetime-to-detection in minutes as outlined in Example 4. NG indicates nodetectable growth for that well in 24 hours and “-” indicates unfilledwells, largely due to the circular shape of the stamped area.

In all, 65 wells out of 131 were positive for growth with an averagetime to detection of 747±11 minutes. Error calculation reflects thestandard error of the mean. The median and mode time-to-detection were740 and 700 minutes respectively and the range was 640 to 1140 minutes.The manufacturer stated time to detection for a single E. coli usingCOLILERT medium is 24 hours in a 100 ml sample and 18 hours using theQUANTI-TRAY® MPN system (IDEXX laboratories; Westbrook, Me.).

Example 6: Fabrication of a Pouch-Like Article and Partitioning of aLiquid Sample Therein

Preparation of a Pouch

The process for preparing a pouch is illustrated in FIGS. 4a-4e . Asubstrate 12 composed of biaxially oriented polypropylene was coatedwith a silicone based pressure sensitive adhesive (adhesive 15, FIG. 4a) as described in Example 1.

A mask (mask 70 of FIG. 4a ) was constructed from a piece ofpolyethylene terephthalate (PET) release liner coated with alow-adhesion fluorosilicone backsize (available from Siliconature USA;Chicago, Ill.). The mask 70 had a 6 cm diameter circular opening 74 anda 1 cm wide gap 76 on one side of the perimeter 72 of the mask. Thelow-adhesion side of the mask 70 was placed onto the adhesive 15 coatedon the substrate 12, as shown in FIG. 4 b.

A secondary coating 18 of glass bubbles (K37 glass bubbles, 3M Company;St. Paul, Minn.) was distributed onto the adhesive using a glass pasturepipette and a silicone bulb. Excess glass bubbles were removed byinverting and tapping the film, resulting in the coated article 200shown in FIG. 4c . The mask 70 was then removed to expose the adhesive15 that had been covered by the mask 70, as shown in FIG. 4d . A coverfilm 20 made from PARAFILM M plastic paraffin film, as described inExample 1, was adhered to the exposed adhesive 15, as described below.

A rubber roller was used to seal the cover film and substrate togetherin the areas where the adhesive was not powder-coated with glassbubbles. This effectively formed a pouch 500 having a circular reservoir502 (FIG. 4e ) with an inoculation port 504 on one side. Adhesionbetween the cover film and the substrate was effectively prevented wherethe glass bubbles had been coated onto the adhesive.

Inoculation and Partitioning of Sample in the Pouch Article.

Test solutions comprising a bacteriological medium with a4-methylumbelliferyl-β-D-glucuronide fluorescent indicator (COLILERTmedium, IDEXX laboratories; Westbrook, Me.) and Escherichia coliAmerican Type Tissue Collection #25922 (EZ-CFU, Microbiologics; St.Cloud, Minn.) were prepared as described in Example 1.

For inoculation the pouch was held upright with the inoculation portpointing upward. 1 ml of test solution was dispensed directly throughthe inoculation port into the pouch (as shown in FIG. 5a ) and wasallowed to settle at the bottom of the pouch.

The inoculated pouch was placed on an aluminum block at a 45° angle anda plastic spreading device (PETRIFILM yeast and mold plate spreader, 3MCompany; St. Paul, Minn.) was manually pressed against the pouch (withmoderate manual pressure) to spread the liquid out and seal the liquidinto a circular (6 cm diameter) chamber (chamber 90 bordered byperimeter seal 92, as shown in FIG. 5b ) disposed between the cover filmand the substrate. Spreading at a 45° angle, although not required,facilitated evacuation of air and uniform filling of the circularchamber as the pouch substrate was pressed against the cover film by thespreader. The spreader forced the cover film against the glass bubbles(not shown) driving the glass bubbles into the adhesive, therebyallowing the adhesive to contact the cover film to form the perimeterseal 92 between the substrate and the cover film.

A plastic 384-well microtiter plate (Untreated black #242764, Nalge NuncInternational; Rochester, N.Y.; tool 50 of FIG. 5c ) was placed on topof the sealed pouch with the openings of the wells facing down and anair press (Model A-0019, Janesville Tool and Manufacturing, Inc.;Milton, Wis.) was used to press the microtiter plate down, deforming thecover film up into the cavities of the microtiter plate and sealing thecover film to the substrate to form the partitioned device 2000 having aplurality of compartments 60 with the liquid sample distributed therein.Each compartment 60 was fluidically isolated from the other compartmentsby one or more seal 62.

Examination of the sealed areas between the wells using astereomicroscope revealed that the glass bubbles had been pushed downinto the adhesive layer allowing the cover film to come into contactwith the pressure sensitive adhesive.

Example 7: Fabrication of an Alternative Pouch-Like Article andPartitioning of a Liquid Sample Therein

Preparation of a Pouch

A pouch-like article was prepared as described in Example 6 with theexception that powdered bacteriological medium (BACTO Trypticase SoyBroth (Becton Dickinson and Company, Franklin Lakes, N.J.) was used asthe secondary coating instead of the glass bubbles.

A rubber roller was used to seal the cover film and the substratetogether in the areas not powder coated with bacteriological medium, asdescribed in Example 6. This effectively formed a circular pouch with aninoculation port along one edge, as described in Example 6. Adhesionbetween the cover film and the substrate was effectively prevented wherethe powdered bacteriological medium had been coated onto the adhesive.

Inoculation and Partitioning of Sample

For inoculation the pouch was held upright with the powder coatedinoculation port pointing upward. 1 ml of test solution (comprising abacteriological medium with a 4-methylumbelliferyl-β-D-glucuronidefluorescent indicator (COLILERT bacteriological medium, IDEXXlaboratories; Westbrook, Me.) and Escherichia coli American Type TissueCollection #25922) was dispensed directly into the pouch and allowed tosettle at the bottom.

The inoculated pouch was sealed using a plastic spreading device(PETRIFILM yeast and mold plate spreader, 3M Company; St. Paul, Minn.)as described in Example 6.

A plastic 384-well microtiter plate (Untreated black #242764, Nalge NuncInternational; Rochester, N.Y.) was used to complete the formation ofthe culture device comprising a plurality of compartments, as describedin Example 6.

Visual inspection of the pouch before pressing and examination of thesealed areas between the wells using a stereoscope after pressingrevealed that the powdered medium was dissolved by the addition of thetest sample, re-exposing the adhesive, and allowing the cover film tocome into contact with the pressure sensitive adhesive coated on thesubstrate.

The stamped culture device was placed in a 37° C. incubator.

Example 8: Growth of an Aerobic Microorganism in Compartments of aStamped Culture Device—Quantitative and Qualitative Analysis

A stamped culture device was prepared according to Example 1, using theculture medium, Heterotrophic Plate Count (HPC for QUANTI-TRAY medium)(IDEXX laboratories; Westbrook, Me.) and Pseudomonas aeruginosa ATCC#15442 (American Type Tissue Collection; Manassas, Va.) as the testmicroorganism.

Culture devices were inoculated and incubated at 37° C. for 24 hours asdescribed in Examples 1-3. Aerobic count (AC) PETRIFILM plates (3MCompany; St. Paul, Minn.) were used as a comparator with 1 ml of thesame test solution having been inoculated onto the PETRIFILM plates,spread, and incubated at 37° C. for 24 hours.

After the 24 h of incubation, the compartments of the stamped culturedevices were assessed for growth of bacteria. A stereomicroscope (ZeissLuminar V12 with Axiocam MRc5) equipped with an excitation source (365nm) and an emission filter (400 nm long pass) was used to observe eachcompartment for fluorescence, described above. The results are shown inTable 4.

TABLE 4 Example 8 PETRIFILM Plates (Growth-positive^(a)) (CFU) 34compartments 24 ^(a)Growth was evidenced by positive fluorescence ineach “growth-positive” compartment of Example 8.

The data demonstrate the growth and enumeration of aerobic bacteria inthe stamped culture device.

Example 9: Growth of an Anaerobic Microorganism in Compartments of aStamped Culture Device—Quantitative and Qualitative Analysis

A stamped culture device was prepared according to Example 1, using theculture medium, 37 g/L Brain Heart Infusion Broth, 5 g/L BACTO YeastExtract (Becton, Dickinson and Company; Franklin Lakes, N.J.), 0.5 g/Lcysteine hydrochloride, 0.5 g/L sodium sulfite, and 0.5 g/L iron sulfate(Sigma Aldrich; St. Louis, Mo.).

Clostridium sporogenes ATCC #5384 (American Type Tissue Collection;Manassas, Va.) was chosen as the microorganism to demonstrate the growthof an anaerobic microorganism in the stamped culture devices inconjunction with an anaerobic atmosphere system.

Culture devices were inoculated and incubated in a sealed box with anactivated GASPAK™ EZ Anaerobe Container System Sachet w/Indicator(Becton, Dickinson and Company; Franklin Lakes, N.J.) at 37° C. for 24hours.

Aerobic count (AC) PETRIFILM plates (3M Company; St. Paul, Minn.) wereused as a comparator with 1 ml of the same test solution having beeninoculated onto the film, spread, and incubated at 37° C. for 24 hoursin the same anaerobic container as the stamped culture devices (Table5).

After the incubation period, compartments in the stamped culture devicethat contained a black precipitate, resulting from the reaction ofhydrogen sulfide produced by Clostridium sporogenes ATCC #5384 with theiron present in the medium, were considered positive for growth (Table5). It is observed that there is good growth of anaerobic bacterium inthe stamped culture device as shown in Table 5. FIG. 6 shows a schematictop view of a stamped culture device 1000 having a plurality ofcompartments 60. The shaded compartments 60 a indicate an observable(e.g., visible color change) reaction with an indicator substance (e.g.,the iron present in the medium of Example 9). The unshaded compartments60 b indicate no observable reaction with the indicator substance.

TABLE 5 Example 9 PETRIFILM Plates (Growth-positive^(a)) (CFU) TestSolution 1 65 compartments 45 Test Solution 2  9 compartments 8^(a)Growth was evidenced by a black precipitate in each“growth-positive” compartment of Example 9.

Example 10: Performance of Secondary Biochemical Test

Stamped culture devices were prepared and inoculated with HPC medium asdescribed in Example 2.1. One set of the devices was inoculated withPseudomonas aeruginosa ATCC #15442 and another set of culture deviceswas inoculated with Escherichia coli ATCC #25922. Both sets wereincubated at incubated 37° C. for 24 hours.

After incubation, the compartments of each culture device were observedunder u.v. illumination to detect growth. Positive fluorescence (growth)was observed in at least several compartments of each culture device(i.e., both Pseudomonas aeruginosa and Escherichia coli samples producedfluorescence-positive compartments.

Representative fluorescence-positive compartments from each culturedevice were pierced with a capillary pipet containing 30% H₂O₂ (SigmaChemical Co.). Some wells were pierced through the (foil) substrate.Others were pierced through the cover film. In all cases,fluorescent-positive compartments in the devices inoculated withPseudomonas aeruginosa produced a vigorous bubbling reaction with theperoxide solution. In contrast, all cases, fluorescent-positivecompartments in the devices inoculated with Escherichia coli did notproduce any observable bubbling reaction when the peroxide solution wasintroduced into the compartments.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material cited herein areincorporated by reference. In the event that any inconsistency existsbetween the disclosure of the present application and the disclosure(s)of any document incorporated herein by reference, the disclosure of thepresent application shall govern. The foregoing detailed description andexamples have been given for clarity of understanding only. Nounnecessary limitations are to be understood therefrom. The invention isnot limited to the exact details shown and described, for variationsobvious to one skilled in the art will be included within the inventiondefined by the claims.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

The invention illustratively described herein suitably may be practicedin the absence of any element(s) not specifically disclosed herein.Thus, for example, in each instance herein any of the terms“comprising”, “consisting essentially of”, and “consisting of” may bereplaced with either of the other two terms. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

1. A device, comprising: a base comprising a substrate, the substratehaving a first major surface; a pressure sensitive adhesive adhered toat least a portion of the first major surface; a polymeric cover filmcoupled to the substrate via the adhesive; wherein the cover film is acomposite film comprising a polymer and a tackifier; wherein thecomposite film comprises ethylene vinyl acetate copolymer, a linearcopolymer of ethylene and a higher alkene, and the tackifier; aplurality of closed compartments disposed between the substrate and thecover film, each compartment of the plurality defined by a seal thatprevents liquid communication with at least one other compartment of theplurality; and an aqueous liquid disposed in two or more of the closedcompartments; wherein the seal is formed by contact between the coverfilm and the pressure-sensitive adhesive.
 2. The device of claim 1,wherein the tackifier is selected from a group consisting of a lowmolecular weight polyisobutene, polyterpenes, amorphous polypropylene,and microcrystalline wax.
 3. The device of claim 1, wherein the coverfilm has an elastic recovery less than or equal to 20%.
 4. The deviceaccording to claim 1, wherein the adhesive is water-insoluble.
 5. Thedevice of claim 1, wherein the first major surface is substantiallyplanar.
 6. The device according to claim 1, wherein the substrate ismade of a material selected from the group consisting of polypropylene,polyurethane, polyethylene, polyester, polyimides, fluoropolymers,polycarbonate, polystyrene, a derivative of any one of the foregoingmaterials, and a combination of any two or more of the foregoingmaterials.
 7. The device according to claim 1, wherein the substratecomprises a metal foil.
 8. The device according to claim 1, wherein thepressure sensitive adhesive is made of material which retains itsadhesive property when in contact with an aqueous liquid.
 9. The deviceaccording to claim 1, wherein the pressure sensitive adhesive issilicone polyurea adhesive.
 10. The device according to claim 1, whereinthe thickness of the pressure sensitive adhesive is at least about 0.02mm.
 11. The device according to claim 1, wherein the thickness of atleast a portion of the cover film is 0.01 mm to 0.5 mm.
 12. The deviceaccording to claim 1, wherein the thickness of at least a portion of thecover film when stretched is 0.01 mm to 0.2 mm.
 13. The device accordingto claim 1, wherein the aqueous liquid comprises a biological sample.14. The device of claim 1, wherein each compartment has a volume of 200nL to 10 ml.
 15. The device of claim 1, wherein the device furthercomprises a spacer element disposed between the substrate and the coverfilm.
 16. The device of claim 1, further comprising a secondary coatingdisposed on at least a portion of the pressure sensitive adhesive. 17.The device of claim 16, wherein the secondary coating consistsessentially of dry powder.
 18. A method for analyzing a liquid samplecomprising: depositing a predefined volume of liquid onto a firstsurface of a substrate such that the liquid sample is disposed betweenthe first surface and a polymeric cover film wherein said first surfaceis coated with water-insoluble pressure sensitive adhesive; wherein thecover film is a composite film comprising a polymer and a tackifier;wherein the composite film comprises ethylene vinyl acetate copolymer, alinear copolymer of ethylene and a higher alkene, and the tackifier;urging an external means against the cover film to bring discreteregions of the cover film in contact with the pressure sensitiveadhesive of the substrate resulting in the partitioning of the liquidinto a plurality of closed compartments disposed between the substrateand the cover film; and conducting a quantitative analysis or aqualitative analysis of at least one closed compartment of theplurality.
 19. The method of claim 18 wherein, prior to urging theexternal means against the cover film, the substrate and/or the coverfilm are substantially flat.
 20. The method according to claim 18,wherein the quantitative analysis comprises enumeration ofmicroorganisms or biomolecules in the sample.